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Ion Total RNA-Seq Kit v2 User Guide (Pub. no. 4476286 Rev. D)

1. Component Een id t ne Reaction Platinum PCR SuperMix High Fidelity 45 uL lon 5 PCR Primer v2 1 uL lon 3 PCR Primer v2 1 uL Total volume 47 uL master mix high fidelity amplification t Include 5 10 excess volume to compensate for pipetting error when preparing i Platinum PCR SuperMix High Fidelity contains a proofreading enzyme for a Transfer 6 uL of cDNA sample to a new PCR tube b Transfer 47 uL of the PCR mix to each 6 uL of cDNA sample C Proceed to step 2 Barcoded Library Component Begs Tor ne Reaction Platinum PCR SuperMix High Fidelity 45 uL lon Xpress RNA 3 Barcode Primer Tul Total volume 46 uL t Include 5 10 excess volume to compensate for pipetting error when preparing master mix Platinum PCR SuperMix High Fidelity contains a proofreading enzyme for high fidelity amplification a Transfer 6 uL of cDNA sample to a new PCR tube b Transfer 46 uL of the PCR mix to each 6 uL of cDNA sample c Add 1 uL of the selected Ion Xpress RNA Seq Barcode BC primer choose from BC01 BC16 to each PCR tube d Proceed to step 2 2 Flick the tube or slowly pipet the solution up and down 5 times to mix well then centrifuge briefly to collect the liquid in the bottom of the tube lon Total RNA Seq Kit v2 User Guide 55 3 Chapter 3 Prepare Small RNA Libraries Construct the small RNA library Purify and size select the amplified DNA 56 3 Run the cDNA s
2. Using a positive control to troubleshoot starting on page 49 A general troubleshooting strategy is to perform the Ion Total RNA Seq Kit v2 procedure using the Small RNA Control human placenta total RNA provided with the kit Use 1 ug of Small RNA Control for the hybridization and ligation procedure You can also use the enrichment procedure included in this guide to enrich small RNA from control RNA then use that as the input for ligation Enrich the samples for small RNA on page 44 Note If you are starting from total RNA the yield will be low we strongly recommend that you use purified or enriched small RNA lon Total RNA Seq Kit v2 User Guide 65 Chapter 3 Prepare Small RNA Libraries Troubleshooting 66 lon Total RNA Seq Kit v2 User Guide Supplemental Information Amplified library construction concepts 67 Hybridization and ligation to the Adaptor Mix 0 0 cece eee eee 67 Reverse transcription and size selection 666s cece eee eee eee 67 Using 2100 expert software to assess whole transcriptome libraries 68 Perform a smear analysis 1 0 0 0 cee eee nee 68 Analyze multiple peaks as one peak 6 0 66 keene 69 Using 2100 expert software to assess small RNA libraries 72 Review the median Size 1 eee ee 72 Perform a smear analysis llle 72 Determine the miRNA library 0 0 ccc 74 Amplified library construction concepts
3. 24 lon Total RNA Seq Kit v2 User Guide Chapter 2 Prepare Whole Transcriptome Libraries Construct the whole transcriptome library Component Pia Tor ne Reaction lon Adaptor Mix v2 2 uL Hybridization Solution 3 uL Total volume 5 uL t Include 5 10 excess volume to compensate for pipetting error when preparing the master mix 2 Add 5 uL of hybridization master mix to 3 uL of fragmented RNA sample Fragmented poly A RNA up to 50 ng Fragmented rRNA depleted total RNA up to 100 ng Note If 50 ng of fragmented poly A RNA or 100 ng rRNA depleted total RNA is recovered after fragmentation we recommend using all fragmented RNA for ligation 3 Slowly pipet the solution up and down 10 times to mix then centrifuge briefly 4 Runthe hybridization reaction in a thermal cycler Temperature Time 65 C 10 min 30 C 5 min 5 Onice add the RNA ligation reagents to the 8 uL hybridization reactions a Combine Component Pune Tor ne Reaction Hybridization reaction 8yL 2X Ligation Buffer 10 uL Ligation Enzyme Mix 2 uL Total volume 20 uL t Include 5 10 excess volume to compensate for pipetting error when preparing the master mix IMPORTANT If the 2X Ligation Buffer contains a white precipitate warm the tube at 37 C for 2 5 minutes or until the precipitate is dissolved 2X Ligation Buffer is very viscous pipet slowly to dispense it accurately b
4. An enzymatic reaction or purification performed after RNase Ill treatment failed Repeat the ligation with more fragmented RNA and run a parallel ligation reaction with fragmented Control RNA Using a positive control to troubleshoot page 17 A general troubleshooting strategy is to perform the lon Total RNA Seq Kit v2 procedure using the WT Control RNA HeLa total RNA provided with the kit Use 500 ng of WT Control RNA for the fragmentation procedure starting on Use 100 ng of fragmented WT Control RNA in the amplified library construction procedure starting on page 24 The expected yields for the WT Control RNA should be the same as the RNA and cDNA yields listed on page 39 40 lon Total RNA Seq Kit v2 User Guide Prepare Small RNA Libraries B WOEIKHOW 2 es ete es Ds doti ut tans hated tots ak ond eri Ute cal dh 42 E Prepare the starting material 2 occ eee 43 Guidelines for obtaining small RNA 2 6 6 e eee nee 43 Assess the amount and quality of small RNA in your total RNA samples 43 Enrich the samples for small RNA 6 006s 44 Assess the quality and quantity of the small RNA enriched sample 48 Determine the input amount 0 een 48 B Construct the small RNA library 06 eens 49 Hybridize and ligate the RNA 0 6 rr 49 Perform reverse transcription 0 66 eee eens 51 Purify and size select the cDNA 0 cece nee eens 52 Amplify the cDN
5. 150 1004 i N Es Regon 1 8 amplified DNA 35 100 150 200 x0 40 s amp 0 100 2000 10360 bp falls within the designated range the ap Se bo Sue datrintion nC Conc pahi Molarity pero Color area under the curve 14 11 1 2 026 27 Le BEEN lon Total RNA Seq Kit v2 User Guide 37 Chapter 2 Prepare Whole Transcriptome Libraries Size distributions and yields 4 amplified DNA falls within the designated range the area under the curve 38 Figure 6 Size distribution of amplified library prepared using poly A RNA from 100 ng WT Control RNA FU 400 100 100 ng WT control 400 500 600 1000 2000 10380 bp olor Conc poli Molarity pmol 1 063 56 12 090 0 lon Total RNA Seq Kit v2 User Guide Chapter 2 Prepare Whole Transcriptome Libraries Size distributions and yields Figure 7 Size distribution of amplified library prepared from 50 ng of rRNA depleted total RNA using the Agilent High Sensitivity DNA Kit 50 ng Hela rRNA depleted 20 of amplified DNA falls within the 3 m m ci zu 400 500 600 1000 2000 10380 bp designated range the amis peel ae Sana v 20 area under the curve 2 937 72 31 149 7 Expected yields The recovery of your experimental RNA will depend on its source and quality The following results are typically seen with Human Brain Reference and HeLa RNAs Note Typical amplifie
6. sssssseeeessseeeeeee ees 26 Purity the CDNA secs bette IERI qe Pee Peut d ed Pee vd eda 27 Amplify the CDNA ous cece bebe cae ecg abe HERR RE a 29 Purify the amplified CDNA sesiet uee i en 30 Assess the yield and size distribution of the amplified DNA 32 Pool barcoded whole transcriptome librarieS ooooooooooomommmomo 33 Determine the library dilution required for template preparation 34 Proceed to template preparation 00 34 Size distributions and yields a n cee eens 35 Typical size distributions 6 cece eens 35 Expected yields it Se A d e be ee e eis 39 Troubleshooting acertada Ve ea Re uas veo es vaa e pates edo en 40 Using a positive control to troublesho0t oooooccoccocncccccccccccccc 40 lon Total RNA Seq Kit v2 User Guide 15 2 Chapter 2 Prepare Whole Transcriptome Libraries Workflow Workflow Fragment the whole transcriptome RNA Start with RNA with ERCC RNA Spike In Control Mix page 17 v Fragment the RNA using RNase Ill page 18 pire v w Purify the fragmented RNA page 19 Fragmented RNA v Assess the yield and size distribution of the fragmented RNA page 21 v Construct the whole transcriptome library oa M Hybridize and ligate the RNA page 24 AAL v Perform reverse transcription RT page 26 v Purify the cDNA page 27 v Amplify the cDNA page 29 v Purify the amplified cDNA page 30 oa Pies v Asse
7. Flick the tube or slowly pipet the solution up and down 5 times to mix well then centrifuge briefly to collect the liquid in the bottom of the tube lon Total RNA Seq Kit v2 User Guide 25 Chapter 2 Prepare Whole Transcriptome Libraries Construct the whole transcriptome library 6 Incubate the 20 uL ligation reactions in a thermal cycler at 30 C according to input type and amount RNA Type idu Me Reaction Time Poly A RNA 1 5 ng 1 hour gt 5 ng 30 min rRNA depleted RNA 10 100 ng 1 hour 100 ng 30 min IMPORTANT Set the temperature of the thermal cycler lid to match the block temperature turn OFF the heated lid or leave the thermal cycler open during the incubation Perform reverse transcription RT Use components from the Ion Total RNA Seq Kit v2 Nuclease Free Water 10X RT Buffer 2 5 mM dNTP Mix Ion RT Primer v2 e 10X SuperScript III Enzyme Mix 1 On ice prepare the RT master mix Component EM Tor ne Reaction Nuclease Free Water 2 uL 10X RT Buffer 4 uL 2 5 mM dNTP Mix 2uL lon RT Primer v2 8yL Total volume 16 uL t Include 5 10 excess volume to compensate for pipetting error when preparing the master mix 2 Incubate the RT master mix with the ligated RNA sample a b Add 16 uL of the RT master mix to each 20 uL ligation reaction Gently vortex the reaction to mix thoroughly then centrifuge the reaction briefly to collect the liquid in t
8. a Leave the Processing Plate on the magnetic stand b Add 150 uL of Wash Solution Concentrate with ethanol to each sample c Incubate the samples at room temperature for 30 seconds 5 Remove the supernatant from the beads a Aspirate and discard the supernatant from the plate b Usea P10 or P20 pipettor to remove any residual liquid IMPORTANT Do not disturb the separated magnetic beads Remove all of the Wash Solution Concentrate from each well 20 lon Total RNA Seq Kit v2 User Guide Assess the yield and size distribution of the fragmented RNA C Chapter 2 Prepare Whole Transcriptome Libraries Fragment the whole transcriptome RNA Air dry the beads at room temperature to remove all traces of ethanol by leaving the Processing Plate on the magnetic stand for 1 2 minutes IMPORTANT Do not overdry the beads overdried beads appear cracked Overdrying significantly decreases elution efficiency 6 Elute the fragmented RNA from the beads a b Remove the Processing Plate from the magnetic stand Add 12 uL of pre warmed 37 C Nuclease Free Water to each sample then mix the Nuclease Free Water and beads by pipetting up and down 10 times Incubate at room temperature for 1 minute Place the Processing Plate on the magnetic stand for 1 minute to separate the beads from the solution Wait for the solution to clear before proceeding to the next step For each sample collect the eluant Use the Qubi
9. 80 Life Technologies Corporation and or its affiliate s warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at www lifetechnologies com termsandconditions If you have any questions please contact Life Technologies at www lifetechnologies com support lon Total RNA Seq Kit v2 User Guide Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Phone 1 760 603 7200 Toll Free in USA 800 955 6288 For support visit lifetechnologies com support or email techsupport dlifetech com technologies lifetechnologies com 21 December 2012
10. Backed by more than 20 years of experience Ambion products provide innovative solutions for specialized RNA applications including RNA Sequencing Life Technologies has combined Ambion RNA isolation and gene expression analysis experience with the speed and simplicity of the Ion Torrent PGM platform to optimize each module in the Ion Total RNA Seq Kit v2 Therefore these products provide the highest possible performance in the RNA Seq workflow lon Total RNA Seq Kit v2 User Guide About This Guide Ambion products and expertise 8 lon Total RNA Seq Kit v2 User Guide Product Information m Purpose of the product 6 eee een ees 9 Preparing barcoded libraries issis ise apir en cee eens 9 m Kit components and storage 6 ee nett enn 10 Ion Total RNA Seq Kit v2 12 Reaction Kit 10 Ion Total RNA Seq Kit v2 48 Reaction Kit 11 HMaterials and equipment required but not provided 0 0000 12 Optional Ion Xpress RNA Seq Barcode 01 16 Kit 12 Required for library preparation 0 12 Materials for whole transcriptome librarieS ooooooooooommmmm m 9 o 13 Materials for small RNA libraries 0 eee ee 13 Purpose of the product Use the Ion Total RNA Seq Kit v2 0 Cat no 4475936 and 4479789 to convert RNA transcripts expressed in a cell or tissue into representative cDNA libraries for strand specific RNA sequencing on the Ion Torrent Personal Genome Machine P
11. E a a aa e rr 61 Plotted size distrib tions bx A A ed CEP PEE 61 Size distributions compared ooocococooo nn 64 Tro bleshootlntg s n anton ets s sut nsus ded cepe EL A ke t hice 65 Using a positive control to troubleshoot 0000 0c eee ee eee eee 65 APPENDIX A Supplemental Information 67 Amplified library construction concepts sssuuusssssslllllsls sss 67 Hybridization and ligation to the Adaptor Mix 0 000 cece cece cece 67 Reverse transcription and size selection 0000 cece ee rr 67 Using 2100 expert software to assess whole transcriptome libraries 0 5 68 Perform a smear analysis 2000 ccc eee eee eee ss e 68 Analyze multiple peaks as one peak 2 0c eee eee eee eee eee 69 Using 2100 expert software to assess small RNA libraries 0 00000 e cece e eee ees 72 Review the median size 6 cece e hh 72 Perform a smear analysis ooococcccoc occ 72 Determine the miRNA library ssssssssssssssssssss ess 74 lon Total RNA Seq Kit v2 User Guide Contents APPENDIX B Safety uoce desc prata ex xat dacs varier pesa eque 75 GhemicaliSatety gt 202A ee a DDR os ral ee ess oa ir Macs dotado dara AO ed da 76 Biological hazard safety n nannan unnan e eee nn 77 Documentation and Support 0 00 cece eee eens 79 OD taINING SDSS ioo dee eed oe eh la Ni eee odes tes e Vou A 79 Obtaining Certifica
12. Gently vortex the Nucleic Acid Binding Beads tube to completely resuspend the magnetic beads b Add 7 uL beads to the wells of the Processing Plate c Add 120 uL Binding Solution Concentrate to each well then mix the Concentrate and beads by pipetting up and down 10 times 2 Bind larger RNA to the beads lon Total RNA Seq Kit v2 User Guide 45 46 Chapter 3 Prepare Small RNA Libraries Prepare the starting material Resuspend 0 5 20 ug of total RNA in 75 uL Nuclease Free Water Transfer 75 uL of each RNA sample to a well with beads of the Processing Plate Set a P200 pipettor at 105 uL Attach a new 200 uL tip to the pipettor then pre wet the new 200 uL tip with 100 ethanol by pipetting the ethanol up and down 3 times Without changing tips add 105 uL of 100 ethanol to each well IMPORTANT While dispensing the ethanol do not force out the last drops Remove the last drop by touching the drop to the well wall Change the tip and repeat steps 2c 2d for the remaining wells only if the tip touches the wall Accurate pipetting of 10076 ethanol is critical for size selection Follow the instructions exactly for best results Seta single or multi channel P200 pipettor at 150 uL Attach new 200 uL tips to the pipettor then mix the suspension in each well thoroughly by pipetting the wells up and down 10 times Note The color of the mixture should be homogeneous after mixing Incubate the samples for 5 minutes
13. Hybridization and ligation to the Adaptor Mix Reverse transcription and size selection The procedures in this protocol are based on Life Technologies Ligase Enhanced Genome Detection LEGenD technology patent pending The RNA samples are hybridized and ligated with the lon Adaptor Mix v2 The Ion Adaptor Mix v2 is a set of oligonucleotides with a single stranded degenerate sequence at one end and a defined sequence required for the Ion Personal Genome Machine PGM System at the other end The Ion Adaptor Mix v2 constrains the orientation of the RNA in the ligation reaction such that hybridization with the Ion Adaptor Mix v2 yields template for sequencing from the 5 end of the sense strand Figure 11 illustrates the downstream emulsion PCR primer alignment and the resulting products of templated sphere preparation for sequencing Figure 11 Strand specific RNA sequence information from lon Total RNA Seq Kit products EA a P1 B The RNA population with ligated adaptors is reverse transcribed to generate single stranded cDNA copies of the fragmented RNA molecules Library generation uses a magnetic bead based size selection process to enrich for library fragments within the desired size range lon Total RNA Seq Kit v2 User Guide 67 Appendix A Supplemental Information Using 2100 expert software to assess whole transcriptome libraries Using 2100 expert software to assess whole transcriptome libraries Perform
14. Kit v2 User Guide Chapter 3 Prepare Small RNA Libraries Typical size distribution Figure 9 Size distribution of non barcoded library prepared from placenta total RNA without enriching small RNA Fu 500 ng placenta total 100 54 amplified DNA 15 50 100 200200 400 50 700 150 bp falls within the designated range the area under the curve Name From bp aa 2 Regon2 86 lon Total RNA Seq Kit v2 User Guide 63 Chapter 3 Prepare Small RNA Libraries Typical size distribution Figure 10 Size distribution of barcoded small RNA library prepared from enriched small RNA FU 58 4 amplified DNA falls within the 30 40 50 0700 Barcoded small RNA i 2000 10380 op of Total Color Conc pai Molarity pencil designated range the n a 47 194 3 area under the curve o mes Size distributions d Size of Size of Barcoded compare Non barcoded 7 Library on the Insert Length Library on the Bioanalyzer Bioanalyzer Instrument Instrument 0 bp 77 bp 85 bp 10 bp 87 bp 95 bp 20 bp 97 bp 105 bp 50 bp 127 bp 135 bp 64 lon Total RNA Seq Kit v2 User Guide Troubleshooting Chapter 3 Prepare Small RNA Libraries Troubleshooting Observation Possible Cause Solution The Agilent software does not calculate one concentration and peak size The software detects multiple peaks in th
15. Plate Take the samples off of the magnetic stand Set a single or multi channel P200 pipettor at 150 uL Attach new 200 uL tips to the pipettor then mix the suspension in each well thoroughly by pipetting the wells up and down 10 times Note The color of the mixture should be homogeneous after mixing Incubate the samples for 5 minutes at room temperature off of the magnetic stand 53 3 Chapter 3 Prepare Small RNA Libraries Construct the small RNA library 5 Remove the supernatant from the beads a Place the Processing Plate on a magnetic stand for 5 minutes to separate the beads from the solution Wait for the solution to clear before proceeding to the next step b Leave the Processing Plate on the magnetic stand then carefully aspirate and discard the supernatant from the plate IMPORTANT Do not disturb the magnetic beads If any beads are aspirated leave 2 3 microliters of supernatant behind 6 Wash the beads with the Wash Solution Concentrate with ethanol a Leave the Processing Plate on the magnetic stand then add 150 uL of Wash Solution Concentrate with ethanol to each sample b Incubate the samples for 30 seconds c Leave the Processing Plate on the magnetic stand then aspirate and discard the supernatant from the plate IMPORTANT Do not disturb the separated magnetic beads Remove all of the Wash Solution Concentrate from each well d Use a P10 or P20 pipettor to remove residual et
16. Remove the last drop by touching the drop to the well wall Change the tip and repeat steps 2b 2c for the remaining wells only if the tip touches the wall Accurate pipetting of 10076 ethanol is critical for best results d Seta single or multi channel P200 pipettor at 150 uL Attach new 200 uL tips to the pipettor then mix the suspension in each well thoroughly by pipetting the wells up and down 10 times Note The color of the mixture should be homogeneous after mixing e Incubate the samples for 5 minutes at room temperature 3 Remove the supernatant from the beads a Place the Processing Plate on a magnetic stand for 5 minutes to separate the beads from the solution Wait for the solution to clear before proceeding to the next step b Leave the Processing Plate on the magnetic stand then transfer the supernatant to a new well on the plate or to a well on a new plate IMPORTANT Do not disturb the magnetic beads If any beads are aspirated leave 2 3 microliters of supernatant behind 4 Bind desired cDNA products to the beads a Remove the Processing Plate from the magnetic stand b Add 35 uL of Nuclease Free Water to the supernatant in the new sample well lon Total RNA Seq Kit v2 User Guide 57 58 Chapter 3 Prepare Small RNA Libraries Construct the small RNA library Set a P100 or P200 pipettor at 35 uL Attach a new 100 or 200 uL tip to the pipettor then pre wet the new 100 or 200 uL tip with 100 ethan
17. at room temperature off of the magnetic stand 3 Remove the supernatant from the beads a Place the Processing Plate on a magnetic stand for 5 minutes to separate the beads from the solution Wait for the solution to clear before proceeding to the next step Leave the Processing Plate on the magnetic stand then transfer the supernatant to a new well on the plate or to a well on a new plate IMPORTANT Do not disturb the magnetic beads If any beads are aspirated leave 2 3 microliters of supernatant behind 4 Bind desired small RNA products to the beads a b Remove the Processing Plate from the magnetic stand Add 30 uL of Nuclease Free Water to the supernatant in the new sample well Set a P1000 pipettor at 570 uL Attach a new 1000 uL tip to the pipettor then pre wet the new 1000 uL tip with 100 ethanol by pipetting the ethanol up and down 3 times Without changing tips add 570 uL of 100 ethanol to each well IMPORTANT While dispensing the ethanol do not force out the last drops Remove the last drop by touching the drop to the well wall Change the tip and repeat steps 4c 4d for the remaining wells only if the tip touches the wall Accurate pipetting of 10076 ethanol is critical for size selection Follow the instructions exactly for best results Gently vortex the Nucleic Acid Binding Beads tube to completely resuspend the magnetic beads lon Total RNA Seq Kit v2 User Guide f g Chapt
18. chemical wastes in a fume hood Ensure use of primary and secondary waste containers A primary waste container holds the immediate waste A secondary container contains spills or leaks from the primary container Both containers must be compatible with the waste material and meet federal state and local requirements for container storage After emptying a waste container seal it with the cap provided Characterize by analysis if necessary the waste generated by the particular applications reagents and substrates used in your laboratory Ensure that the waste is stored transferred transported and disposed of according to all local state provincial and or national regulations IMPORTANT Radioactive or biohazardous materials may require special handling and disposal limitations may apply lon Total RNA Seq Kit v2 User Guide Appendix B Safety Biological hazard safety Biological hazard safety AN lon Total RNA Seq Kit v2 User Guide WARNING BIOHAZARD Biological samples such as tissues body fluids infectious agents and blood of humans and other animals have the potential to transmit infectious diseases Follow all applicable local state provincial and or national regulations Wear appropriate protective equipment which includes but is not limited to protective eyewear face shield clothing lab coat and gloves All work should be conducted in properly equipped facilities using the appropriate safety e
19. ethanol Store the solution at room temperature Ifyou see a white precipitate in the Binding Solution Concentrate warm the solution at 37 C then shake the solution to dissolve any precipitate before use e Incubate the Nuclease Free Water at 37 C for 25 minutes lon Total RNA Seq Kit v2 User Guide Chapter 3 Prepare Small RNA Libraries 3 Construct the small RNA library Note To reduce the chance of cross contamination we strongly recommend sealing unused wells on the Processing Plate with MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 You can also skip a row between sample rows IMPORTANT Accurate pipetting of bead cleanup reagents is critical for best results 1 Prepare beads for each sample a Gently vortex the Nucleic Acid Binding Beads tube to completely resuspend the magnetic beads b Add 7 uL beads to wells on the Processing Plate c Add 140 uL Binding Solution Concentrate to each well then mix the Concentrate and beads by pipetting up and down 10 times 2 Bind larger amplified DNA to the beads a Transfer each 53 uL PCR reaction to a well with beads of the Processing Plate b Set a P200 pipettor at 110 uL Attach a new 200 uL tip to the pipettor then pre wet the new 200 uL tip with 100 ethanol by pipetting the ethanol up and down 3 times c Without changing tips add 110 uL of 100 ethanol to each well IMPORTANT While dispensing the ethanol do not force out the last drops
20. final molar concentration of the pooled library is the same for each diluted library For example if you dilute each library to 30 nM the concentration of the pooled library is 30 nM Use the final molar concentration to determine the Template Dilution Factor You can also determine the molar concentration of the pooled libraries with the Agilent DNA 1000 Kit or the Agilent High Sensitivity DNA Kit see Assess the yield and size distribution of the amplified DNA on page 59 and Using 2100 expert software to assess small RNA libraries on page 72 Determine the library dilution required for template preparation 60 For template preparation using an appropriate Ion template preparation kit determine the library dilution that gives 210 x 106 molecules of template per 20 uL 210 x 106 is the recommended library input for Whole Transcriptome libraries Use a conversion factor of 8 3 nM 5 x 10 molecules uL and the following formula Template Dilution Factor Library concentration in nM x 5 x 109 molecules uL 8 3 nM x volume per template preparation reaction inuL 210 x 106 molecules For the volume per template preparation reaction refer to the specific user guide for the appropriate Ion template preparation kit If your library concentration is gt 20 nM serially dilute 1 10 or 1 100 until your library concentration is X20 nM This step helps prevent a Template Dilution Factor of several thousands Exampl
21. minutes at room temperature off of the magnetic stand 3 Remove the supernatant from the beads a Place the Processing Plate on a magnetic stand for 5 minutes to separate the beads from the solution Wait for the solution to clear before proceeding to the next step Leave the Processing Plate on the magnetic stand then transfer the supernatant to a new well on the plate or to a well on a new plate IMPORTANT Do not disturb the magnetic beads If any beads are aspirated leave 2 3 microliters of supernatant behind 4 Bind desired cDNA products to the beads lon Total RNA Seq Kit v2 User Guide a b Remove the Processing Plate from the magnetic stand Add 72 uL of Nuclease Free Water to the supernatant in the new sample well Set a P100 or P200 pipettor at 78 uL Attach a new 100 uL or 200 uL tip to the pipettor then pre wet the new 100 or 200 uL tip with 100 ethanol by pipetting the ethanol up and down 3 times Without changing tips add 78 uL of 100 ethanol to each well IMPORTANT While dispensing the ethanol do not force out the last drops Remove the last drop by touching the drop to the well wall Change the tip and repeat steps 4c 4d for the remaining wells only if the tip touches the wall Accurate pipetting of 10076 ethanol is critical for best results Gently vortex the Nucleic Acid Binding Beads tube to completely resuspend the magnetic beads Add 7 uL beads to the wells of the Processing
22. prepare the PCR mix according to the non barcoded or barcoded library tables IMPORTANT Use the appropriate primers Non barcoded Library Component piece Tor t ne Reaction Platinum PCR SuperMix High Fidelity 45 uL lon 5 PCR Primer v2 1 uL lon 3 PCR Primer v2 1 uL Total volume 47 uL t Include 5 10 excess volume to compensate for pipetting error when preparing master mix i Platinum PCR SuperMix High Fidelity contains a proofreading enzyme for high fidelity amplification a Transfer 6 uL of cDNA sample to a new PCR tube b Transfer 47 uL of the PCR mix to each 6 uL of cDNA sample c Proceed to step 2 Barcoded Library Component RE i ne Reaction Platinum PCR SuperMix High Fidelity 45 uL lon Xpress RNA 3 Barcode Primer 1 uL Total volume 46 uL t Include 5 10 excess volume to compensate for pipetting error when preparing master mix i Platinum PCR SuperMix High Fidelity contains a proofreading enzyme for high fidelity amplification a Transfer 6 uL of cDNA sample to a new PCR tube b Transfer 46 uL of the PCR mix to each 6 uL of cDNA sample lon Total RNA Seq Kit v2 User Guide 29 Chapter 2 Prepare Whole Transcriptome Libraries Construct the whole transcriptome library c Add 1 uL of the selected Ion Xpress RNA Seq Barcode BC primer choose from BC01 BC16 to each PCR tube d Proceed to step 2 2 F
23. the chance of cross contamination we strongly recommend sealing unused wells on the Processing Plate with MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 You can also skip a row between sample rows 30 lon Total RNA Seq Kit v2 User Guide Chapter 2 Prepare Whole Transcriptome Libraries Construct the whole transcriptome library IMPORTANT Accurate pipetting of bead cleanup reagents is critical for best results 1 Prepare beads for each sample a Gently vortex the Nucleic Acid Binding Beads tube to completely resuspend the magnetic beads b Add 5 uL beads to wells on the Processing Plate c Add 180 uL Binding Solution Concentrate to each well then mix the Concentrate and beads by pipetting up and down 10 times 2 Bind the amplified cDNA to the beads a Transfer 53 uL of each amplified cDNA sample to a bead containing well on the Processing Plate b Set a P200 pipettor at 130 uL Attach a new 200 uL tip to the pipettor then pre wet the new 200 uL tip with 100 ethanol by pipetting the ethanol up and down 3 times c Without changing tips add 130 uL of 100 ethanol to each well IMPORTANT While dispensing the ethanol do not force out the last drops Remove the last drop by touching the drop to the well wall Change the tip and repeat steps 2b 2c for the remaining wells only if the tip touches the wall Accurate pipetting of 10076 ethanol is critical for best results d Seta single or multi ch
24. total RNA sample Use the and quality of small NanoDrop Spectrophotometer and the Agilent 2100 Bioanalyzer instrument with RNA in your total the Agilent RNA 6000 Nano Kit and the Agilent Small RNA Kit RNA samples 1 Quantitate the amount of RNA in the sample using the NanoDrop Spectrophotometer Note If you used the mirVana miRNA Isolation Kit the mirVana PARIS Kit or the PureLink miRNA Isolation Kit to isolate small RNA from samples you can skip to Assess the quality and quantity of the small RNA enriched sample on page 48 2 Determine the quality of the small RNA in your sample Note For instructions on using the software refer to the Agilent 2100 Bioanalyzer Expert User s Guide Pub no G2946 9000 a Dilute the RNA to 50 100 ng uL b Run 1 uL of diluted RNA on the Agilent 2100 Bioanalyzer instrument with the Agilent RNA 6000 Nano chip to determine the concentration of total RNA Follow the manufacturer s instructions for performing the assay c Using the 2100 expert software determine the mass of total RNA in the sample and save the mass of total RNA for step 3c to calculate the miRNA content lon Total RNA Seq Kit v2 User Guide 43 Chapter 3 Prepare Small RNA Libraries Prepare the starting material Enrich the samples for small RNA 44 d Using the 2100 expert software review the RNA Integrity Number RIN The highest quality library mapping statistics are obtained from input RNA w
25. troubleshoot 00 000 c cece eee eee eee eae 40 CHAPTER 3 Prepare Small RNA Libraries 0o ooo ooo 41 Workflow sessi ere xe mex aue v nd d e Rae d Ie e d Rd Ro iere one 42 Prepare the starting material 020 se 43 Guidelines for obtaining small RNA ssssssssssssse eee eee 43 Assess the amount and quality of small RNA in your total RNA samples 43 Enrich the samples for small RNA 00000 c cece eee eee e eee 44 Assess the quality and quantity of the small RNA enriched sample 48 Determine the input amount 0000 rr 48 Construct the small RNA library 0 000 c eect n 49 Hybridize and ligate the RNA 020s 49 Perform reverse transcription 0000 0c eee eee eee eee e eee e teens 51 Purify and size select the cDNA 00 cece eee eee eee eee eeee 52 Amplify the cDNA az ELDER dete eee ee Scere EUER at A 54 Purify and size select the amplified DNA lliliiiiiiuisissslslsllsslslll lese 56 Assess the yield and size distribution of the amplified DNA ssullllllslslsss 59 Pool barcoded small RNA libraries ssssssssssssssese n nt t eee tne eee eee 60 Determine the library dilution required for template preparation 00000 ee 60 Proceed to template preparation 60 0c cee eee eee eee eee eee ees 61 Typical size distribution a e E
26. 0 of DNA in the range gt 60 We recommend that you perform another round of purification on the amplified DNA using components from the Magnetic Bead Cleanup Module 1 Bring the sample volume to 53 uL with Nuclease Free Water 2 Follow the steps in Purify the cDNA on page 27 Pool barcoded whole transcriptome libraries Note If you are not pooling libraries skip this section and proceed to Determine the library dilution required for template preparation on page 34 Determine the molar concentration nM of each of the barcoded cDNA libraries with the Agilent DNA 1000 Kit or the Agilent High Sensitivity DNA Kit Note 50 1000 bp size range is typically used to determine library concentration If necessary adjust the range to include all the library peaks Dilute each barcoded cDNA library to the same molar concentration nM For example if you have 3 different barcoded libraries that are 45 55 65 nM choose a concentration that is equal to or lower than the lowest concentration of the three libraries such as 30 nM Dilute all or part of the library to 30 nM Mixan equal volume of each diluted library to prepare a pool of the barcoded libraries The final molar concentration of the pooled library is the same for each diluted library For example if you dilute each library to 30 nM the concentration of the pooled library is 30 nM Use the final molar concentration to determine the Template Di
27. 0 300 bp and analysis 86 106 bp size range The desired size range for miRNA ligation products is 86 106 bp 1 In the 2100 expert software select View Setpoints File Context Yiew Electropherogram Windows Help Data 5 Gel Electrophe bul Electropherogram Context Bar Assay Properties ts Treo View Ira 5 72 lon Total RNA Seq Kit v2 User Guide Appendix A Supplemental Information Using 2100 expert software to assess small RNA libraries 2 On the Global tab select Advanced settings Local Global Advanced M Collapse 3 In the Sample Setpoints section of the Advanced settings select the Perform Smear Analysis checkbox then double click Table Sample Setpoints Alignment Align to Upper Marker x Align to Lower Marker x Quantitation Concentration of Upper 2 1 Concentration of Lower 4 2 Sizing Standard Curve Point to Point Smear Analysis Perform Smear Analysis X Regions Table 4 Setthe smear regions in the Smear Regions dialog box a Click Add then enter 50 bp and 300 bp for the lower and upper limits respectively b Click Add enter 86 bp and 106 bp then click OK Smear Regions Global Setpoints From bp To bp Name Color gt 50 300 Ex 1 2 86 106 5 Select the Region Table tab Results Peak Table Region Table Legend 6 In the Region Table review the area values for each of the si
28. 2 10X RNase Ill Reaction Buffer 1 uL 3 RNase Ill 1 uL Total volume 10 12 uL IMPORTANT To reduce fragmentation variability accurately pipet 1 uL of 10X RNase III Reaction Buffer and 1 uL of RNase III to each sample Do not make a master mix that contains only 10X RNase III Reaction Buffer and RNase III lon Total RNA Seq Kit v2 User Guide Purify the fragmented RNA Chapter 2 Prepare Whole Transcriptome Libraries Fragment the whole transcriptome RNA 2 Flick the tube or pipet up and down 5 times to mix then centrifuge briefly to collect the liquid in the bottom of the tube 3 Incubate the reaction in a thermal cycler at 37 C according to library and input quantity RNA Type Amount Reaction Time Poly A RNA 1 to lt 100 ng 3 min 100 500 ng 10 min rRNA depleted RNA 10 to lt 100 ng 3 min 100 500 ng 10 min Total RNA 500 ng 10 min 4 Immediately after the incubation add 20 uL of Nuclease Free Water then place the fragmented RNA on ice IMPORTANT Proceed immediately to Purify the fragmented RNA or leave the fragmented RNA on ice for less than 1 hour Required materials from the Magnetic Bead Cleanup Module Wash Solution Concentrate Binding Solution Concentrate Nucleic Acid Binding Beads Processing Plate e Nuclease Free Water Other materials and equipment 100 ethanol or 200 proof absolute ethanol ACS grade or higher quality Magnetic stand for 96 well plates Lif
29. 6 and 4369510 Optional RiboMinus Eukaryote System v2 Life Technologies A15027 Optional RiboMinus Plant Kit for RNA Seq Life Technologies A1083808 Item Source Agilent RNA 6000 Nano Kit Agilent 5067 1511 Agilent Small RNA Kit Agilent 5067 1548 Optional mirVana miRNA Isolation Kit 40 purifications Life Technologies AM1560 Optional mirVana PARIS 40 purification systems Life Technologies AM1556 Optional PureLink miRNA Isolation Kit 25 preps Life Technologies K1570 01 lon Total RNA Seq Kit v2 User Guide 13 1 Chapter 1 Product Information Materials and equipment required but not provided 14 lon Total RNA Seq Kit v2 User Guide Prepare Whole Transcriptome Libraries WOPKAOW ite Sa rae ak diced e Ped I RE bebe aa hatia a a 16 Fragment the whole transcriptome RNA 0 066 17 Guidelines for RNA sample type and amount 6 000 e cee eee eee 17 ERCC RNA Spike In Control Mixes 0 000000 17 Fragment the RNA using RNase IT 2 666 18 Purify the fragmented RNA 06 cee eee eens 19 Assess the yield and size distribution of the fragmented RNA 21 Typical results of fragmentation of whole transcriptome RNA 22 Construct the whole transcriptome libraty ooooooooommmcorrrommm 24 Hybridize and ligate the RNA 0 cece een 24 Perform reverse transcription RT
30. A cuota cee bed iier e riipi e Daehn AUR ondes 54 Purify and size select the amplified DNA 0 000 cece eee eee 56 Assess the yield and size distribution of the amplified DNA 59 BW Pool barcoded small RNA libraries 60 EB Determine the library dilution required for template preparation 60 m Proceed to template preparation 6 ne 61 B Typical size distribution isssssssseeee eee nes 61 Plotted size distributions 0 rr 61 Size distributions compared 66 eee eens 64 M Troubleshooting ors a its BS UES bes ay dee a ES 65 Using a positive control to troubleshoot 0 65 lon Total RNA Seq Kit v2 User Guide 41 2 Chapter 3 Prepare Small RNA Libraries Workflow Workflow Prepare the starting material Assess the amount and quality of small RNA in your total RNA samples page 43 Total RNA Small RNA v RIN 9 3 Enrich the samples for small RNA page 44 v Assess the quality and quantity of the small RNA enriched sample page 48 v Determine the input amount page 48 Construct the small RNA library en i Hybridize and ligate the RNA page 49 PONDUS v Perform reverse transcription page 51 v Purify and size select the cDNA page 52 v Amplify the cDNA page 54 v Purify and size select the amplified DNA page 56 v 5 PCR Primer Assess the yield and size distribution of the amplified DNA page 59 Y Y PCR Primer Pool barcoded sm
31. A reaction to a bead containing well on the Processing Plate b Set a P200 pipettor at 150 uL Attach a new 200 uL tip to the pipettor then pre wet the new 200 uL tip with 100 ethanol by pipetting the ethanol up and down 3 times c Without changing tips add 150 uL of 10076 ethanol to each well IMPORTANT While dispensing the ethanol do not force out the last drops Remove the last drop by touching the drop to the well wall Change the tip and repeat steps 2b 2c for the remaining wells only if the tip touches the wall Accurate pipetting of 10076 ethanol is critical for best results d Set a single or multi channel P200 pipettor at 150 uL Attach new 200 uL tips to the pipettor then mix the suspension in each well thoroughly by pipetting the wells up and down 10 times Note The color of the mixture should be homogeneous after mixing e Incubate the samples for 5 minutes at room temperature off of the magnetic stand 3 Remove the supernatant from the beads a Place the Processing Plate on a magnetic stand for 5 6 minutes to separate the beads from the solution Wait for the solution to clear before proceeding to the next step b Leave the Processing Plate on the magnetic stand then aspirate and discard the supernatant from the plate IMPORTANT Do not disturb the magnetic beads If any beads are aspirated leave 2 3 microliters of supernatant behind 4 Wash the beads with Wash Solution Concentrate with ethanol
32. ANT Accurate pipetting of bead cleanup reagents is critical for best results 1 Prepare beads for each sample a Gently vortex the Nucleic Acid Binding Beads tube to completely resuspend the magnetic beads b Add 7 uL beads to the wells of the Processing Plate c Add 140 uL Binding Solution Concentrate to each well then mix the Concentrate and beads by pipetting up and down 10 times 2 Bind larger cDNA products to the beads a Transfer each 40 uL RT reaction to a well with beads of the Processing Plate b Set a P200 pipettor at 120 uL Attach a new 200 uL tip to the pipettor then pre wet the new 200 uL tip with 100 ethanol by pipetting the ethanol up and down 3 times lon Total RNA Seq Kit v2 User Guide C e Chapter 3 Prepare Small RNA Libraries 3 Construct the small RNA library Without changing tips add 120 uL of 100 ethanol to each well IMPORTANT While dispensing the ethanol do not force out the last drops Remove the last drop by touching the drop to the well wall Change the tip and repeat steps 2b 2c for the remaining wells only if the tip touches the wall Accurate pipetting of 100 ethanol is critical for best results Seta single or multi channel P200 pipettor at 150 uL Attach new 200 uL tips to the pipettor then mix the suspension in each well thoroughly by pipetting the wells up and down 10 times Note The color of the mixture should be homogeneous after mixing Incubate the samples for 5
33. Binding Solution Concentrate warm the solution at 37 C then shake the solution to dissolve any precipitate before use e Incubate the Nuclease Free Water at 37 C for 25 minutes Note To reduce the chance of cross contamination we strongly recommend sealing unused wells on the Processing Plate with MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 You can also skip a row between sample rows IMPORTANT Accurate pipetting of bead cleanup reagents is critical for best results 1 Prepare beads for each sample a Gently vortex the Nucleic Acid Binding Beads tube to completely resuspend the magnetic beads b Add 5 uL beads to wells on the Processing Plate c Add 120 uL Binding Solution Concentrate to each well then mix the Binding Solution Concentrate and beads by pipetting up and down 10 times 2 Bind the cDNA to the beads a Add 60 uL of Nuclease Free Water to each of the 40 uL RT reaction b Transfer each 100 uL RT reaction to a bead containing well on the Processing Plate c Seta P200 pipettor at 125 uL Attach a new 200 uL tip to the pipettor then pre wet the new 200 uL tip with 100 ethanol by pipetting the ethanol up and down 3 times lon Total RNA Seq Kit v2 User Guide 27 28 Chapter 2 Prepare Whole Transcriptome Libraries Construct the whole transcriptome library d Without changing tips add 125 uL of 100 ethanol to each well IMPORTANT While dispensing the ethanol do not force out
34. GM System and the Ion Proton System This user guide supports library preparation for up to 200 base read sequencing For whole transcriptome libraries follow the procedures in Chapter 2 on page 15 e For small RNA libraries follow the procedures in Chapter 3 on page 41 Pre pa ring The Ion Total RNA Seq Kit v2 0 supports barcoded library preparation to enable barcoded libraries sequencing of multiple samples in a single multiplexed sequencing run To prepare arcoded libraries replace the adaptors in the kit with adaptors from the Ion Xpress RNA Seq Barcode 01 16 Kit Cat no 4475485 M lon Total RNA Seq Kit v2 User Guide 9 Chapter 1 Product Information Kit components and storage Kit components and storage lon Total RNA Seq Kit v2 12 Reaction Sufficient reagents are supplied in the Ion Total RNA Seq Kit v2 12 Reaction Kit Cat no 4475936 to prepare cDNA libraries from 12 samples for sequencing analysis with the Ion PGM System Kit Box Components Cap Color Quantity Volume Storage lon RNA Seq Core Kit Nuclease Free Water Clear 2 1 75 mL 15 C to 30 C v2 12 Reaction Kit room temperature Part no 4474906 10X RNase Ill Reaction Buffer Red 1 20 pL 30 C to 10 C RNase III Red 1 20 uL Hybridization Solution Green 1 40 uL 2X Ligation Buffer Green 1 150 uL Ligation Enzyme Mix Green 1 30 uL 10X RT Buffer Yellow 1 56 uL 2 5 mM dNTP Mix Wh
35. II Enzyme Mix 1 On ice prepare the RT master mix with the ligated RNA sample Component Tene Mele ne Reaction Nuclease Free Water 2 uL 10X RT Buffer 4 uL 2 5 mM dNTP Mix 2 uL lon RT Primer v2 8 uL Total volume 16 uL t Include 5 1096 excess volume to compensate for pipetting error when preparing the master mix 2 Incubate the RT master mix with the ligated RNA sample a Add 16 uL of RT master mix to each 20 uL ligation reaction b Pipet up and down 5 times to mix then centrifuge briefly to collect the liquid in the bottom of the tube C Incubate in a thermal cycler with a heated lid at 70 C for 10 minutes then snap cool on ice 3 Perform the reverse transcription reaction a Add 4 uL of 10X SuperScript III Enzyme Mix to each ligated RNA sample b Gently vortex to mix thoroughly then centrifuge briefly c Incubate in a thermal cycler with a heated lid at 42 C for 30 minutes STOPPING POINT The cDNA can be stored at 30 C to 10 C for 2 weeks stored at 86 C to 68 C for long term storage or used immediately lon Total RNA Seq Kit v2 User Guide 51 3 Chapter 3 Prepare Small RNA Libraries Construct the small RNA library Purify and size select the cDNA 52 Use the Magnetic Bead Cleanup Module twice with the same sample to size select the desired cDNA products During the first bead binding magnetic beads capture larger cDNA species such as tRNA and rRNA During the second bindin
36. RN III Red 1 60 uL Part no 4479687 ae H Hybridization Solution Green 1 170 uL 2X Ligation Buffer Green 1 650 uL Ligation Enzyme Mix Green 1 110 uL 10X RT Buffer Yellow 1 224 uL 2 5 mM dNTP Mix White 1 120 uL 10X SuperScript Ill Enzyme Yellow 1 224 uL Mix Platinum PCR SuperMix Blue 2 1800 uL High Fidelity WT Control RNA 1 ug uL Clear 1 50 uL HeLa total RNA Small RNA Control 1 ug uL Purple 1 10 uL human placenta total RNA lon RNA Seq Primer lon Adaptor Mix v2 Green 1 tube 120 uL 30 C to 10 C Set v2 48 Reaction E Kit Part no 4475482 lon RT Primer v2 Yellow 1 tube 416 uL lon 5 PCR Primer v2 White 1 tube 80 uL lon 3 PCR Primer v2 Blue 1 tube 80 uL lon RNA Seq Core Kit 10X RNase IIl Reaction Buffer Red 1 60 uL 30 C to 10 C v2 48 Reaction Kit Part no 4479687 Magnetic Bead Processing Plate 1 15 C to 30 C E bad Binding Solution Concentrate 1 bottle 11 mL room temperature Wash Solution Concentrate 1 bottle 11 mL Nucleic Acid Binding Beads Clear 1 tube 0 7 mL 2 C to 8 C IMPORTANT Do not freeze Nuclease Free Water 1 bottle 10 mL 15 C to 30 C room temperature t Life Technologies has validated this protocol using this specific material Substitution may adversely affect performance lon Total RNA Seq Kit v2 User Guide 11 1 Chapter 1 Product Information Materials and equipment required but not provided Materials and equipment required but not provided Optional lon Xpr
37. System User Bulletin Pub no 4478119 34 lon Total RNA Seq Kit v2 User Guide Chapter 2 Prepare Whole Transcriptome Libraries Size distributions and yields Size distributions and yields Typical size The highest quality libraries have less than 50 amplified DNA between 25 160 bp distributions Figure 3 Molar concentration and size distribution of amplified library prepared from HeLa poly A RNA FU 40 11 amplified DNA falls within the designated range the 77 area under the curve 2 2 Thelibrary concentration Cs 224 7 nM lon Total RNA Seq Kit v2 User Guide 35 Chapter 2 Prepare Whole Transcriptome Libraries 36 Size distributions and yields Figure 4 Molar concentration and size distribution of amplified library prepared from HeLa rRNA depleted total RNA FU 10 amplified DNA falls within the The library designated range the ep 2 A 4 00 concentration area under the curve 2 fe 296 36 Ey is 224 1 nM lon Total RNA Seq Kit v2 User Guide Chapter 2 Prepare Whole Transcriptome Libraries Size distributions and yields Figure 5 Size distribution of amplified library prepared using 5 ng of poly A RNA and isolated using the Dynabeads mRNA DIRECT Micro Kit Refer to Poly A selection from 100 ng 1ug input Total RNA samples protocol in the Dynabeads mRNA DIRECT Micro Kit User Guide FU Sng Poly A RNA 3004 Ew 200 4
38. USER GUIDE ion torrent 6x AOXO ty Life technologies lon Total RNA Seq Kit v2 for use with lon Personal Genome Machine PGM System lon Proton System Catalog Number 4475936 and 4479789 Publication Number 4476286 Revision D For research use only Not for use in diagnostic procedures technologies For Research Use Only Not for use in diagnostic procedures The information in this guide is subject to change without notice DISCLAIMERS LIFE TECHNOLOGIES CORPORATION AND OR ITS AFFILIATE S DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY FITNESS FOR A PARTICULAR PURPOSE OR NON INFRINGEMENT TO THE EXTENT ALLOWED BY LAW IN NO EVENT SHALL LIFE TECHNOLOGIES AND OR ITS AFFILIATE S BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF TRADEMARKS The trademarks mentioned herein are the property of Life Technologies Corporation and or its affiliate s or their respective owners Bioanalyzer and Agilent are registered trademarks of Agilent Technologies Inc TagMan is a registered trademark of Roche Molecular Systems Inc and is used under permission and license NanoDrop is a registered trademark of NanoDrop Technologies LLC SpeedV
39. a smear Perform a smear analysis to quantify the percentage of DNA in the 25 160 bp size analysis range 1 In the 2100 expert software select View gt Setpoints File Context View Electropherogram Windows Help Data 5 Gel ie Electrophe hu Electropherogram Context Bar Assay Properties Ts Tree View Eje 2 2 On the Global tab select Advanced settings Wf Electropherog FU Local Global Advanced M Collapse 3 In the Sample Setpoints section of the Advanced settings select the Perform Smear Analysis checkbox then double click Table Sample Setpoints Alignment Align to Upper Marker x Align to Lower Marker x Quantitation Concentration of Upper 2 1 Concentration of Lower 4 2 Sizing Standard Curve Point to Point Smear Analysis Perform Smear Analysis X Regions Table 4 Setthe smear regions in the Smear Regions dialog box Click Add then enter 25 bp and 160 bp for the lower and upper limits respectively These settings are used to determine the percentage of total product that is 25 160 bp in length Smear Regions Local Setpoints From bp To bp gt 25 160 Delete Add 5 Select the Region Table tab Results Peak Table Region Table Legend 68 lon Total RNA Seq Kit v2 User Guide Using 2100 expert software to assess whole transcriptome libraries Appendix A Supplemental Information 6 In t
40. ac is a registered trademark of Thermo Fisher Scientific 2012 Life Technologies Corporation All rights reserved Contents About THIS Guide cios el 7 Revision HIStory s ea hes sede dea o ex cde tee eget med S eu EI NUR d ceti c 7 Other lon library preparation kits and guides 000 c eee ceee cette e eens 7 Ambion products and expertise 2 0 00 c ccc cece cece tenn hn 7 CHAPTER 1 Product Information s seus 9 Purpose of the product 02 cc cece cece rrr 9 Preparing barcoded libraries cece cece eee cece eaee 9 Kit components and storage 2 2 ene ence eee nn 10 lon Total RNA Seq Kit v2 12 Reaction Kit 00 0 cee eee ees 10 lon Total RNA Seq Kit v2 48 Reaction Kit 0 0 ccc ees 11 Materials and equipment required but not provided 0000 00 cece eee eee eee eens 12 Optional lon Xpress RNA Seq Barcode 01 16 Kit 0 2 0 0 ccc ccc eee nee 12 Required for library preparation 000 cece eee eee eee aes 12 Materials for whole transcriptome libraries 0000 0c eee eee eee eens 13 Materials for small RNA libraries 000 00 eee eees 13 Hi CHAPTER 2 Prepare Whole Transcriptome Libraries 15 Worktlows csi esee eet nt on hoe tee etd etie al e PRENDA aber alta te eds 16 Fragment the whole transcriptome RNA 2 200 cee eee eee eee e eee aee 17 Guidelines for RNA sample type a
41. all RNA libraries page 60 v A sequence P1 sequence Determine the library dilution required for template preparation page 60 v Prepare templated lon Sphere particles for sequencing on the lon POM System Refer to the end user documentation for the lon OneTouch System a Pub no 4478372 OC 0 42 lon Total RNA Seq Kit v2 User Guide Chapter 3 Prepare Small RNA Libraries Prepare the starting material Prepare the starting material Preparing the starting material involves the following procedures Assess the amount and quality of small RNA in your total RNA samples page 43 i Enrich the samples for small RNA page 44 i Assess the quality and quantity of the small RNA enriched sample page 48 Guidelines for For this protocol the total RNA must contain the small RNA fraction microRNA or obtaining small miRNA 10 40 nt For optimal results use RNA that has been size selected for small RNA RNA We recommend the following products RNA source Best results are obtained using high quality total RNA with an RNA integrity number RIN gt 6 as your starting material e RNA isolation kits Use the mirVana miRNA Isolation Kit or the mirVana PARIS Kit to isolate total RNA that includes the small RNA fraction or small RNA from tissue or cells Use the PureLink miRNA Isolation Kit to isolate small RNA from tissues or cells Assess the amount Before you prepare the library determine the quality of the
42. ample check the profile of the poly A RNA on an Agilent 2100 Bioanalyzer instrument Guidelines for using rRNA depleted total RNA To prepare rRNA depleted total RNA from 1 5 ug total RNA we recommend that you remove rRNA using the RiboMinus Eukaryote System v2 Cat no A15026 100 ng 1 ug total RNA we recommend that you remove rRNA using the Low Input RiboMinus Eukaryote System v2 Cat no A15027 Guidelines for using total RNA Best results are obtained when using RNA with an RNA integrity number RIN greater than 7 FirstChoice Total RNA provides high quality intact RNA isolated from a variety of sources Quantitate the amount of RNA in the sample using the NanoDrop Spectrophotometer We strongly recommend that you add ERCC RNA Spike In Control Mixes to the input total RNA before RNA depletion or poly A selection for whole transcriptome library generation The ERCC RNA Spike In Control Mixes provide a set of external RNA controls that enable performance assessment of a variety of technology platforms used for gene expression experiments Add one Spike In Control Mix to each RNA sample and run these samples on your platform compare the Spike In Control Mix data to known lon Total RNA Seq Kit v2 User Guide 17 Chapter 2 Prepare Whole Transcriptome Libraries Fragment the whole transcriptome RNA Fragment the RNA using RNase III 18 Spike In Control Mix concentrations and ratios to assess the dy
43. amples in a thermal cycler Stage Temp Time Hold 94 C 2 min Cycle 2 cycles 94 C 30 sec 50 C 30 sec 68 C 30 sec Cycle 14 cycles 94 C 30 sec 62 C 30 sec 68 C 30 sec Hold 68 C 5 min Use the Magnetic Bead Cleanup Module twice with the same sample to size select the desired cDNA products During the first round of bead binding magnetic beads capture larger cDNA species such as tRNA and rRNA During the second round of bead binding and with increased ethanol concentration desired cDNA products miRNA and other small RNA in the supernatant re bind to the magnetic beads After washing the beads the desired cDNA products are eluted with pre warmed 37 C Nuclease Free Water Required materials from the Magnetic Bead Cleanup Module Wash Solution Concentrate Binding Solution Concentrate Nucleic Acid Binding Beads Processing Plate e Nuclease Free Water Other materials and equipment 100 ethanol or 200 proof absolute ethanol ACS grade or higher quality Optional Orbital shaker for 96 well plates Magnetic stand for 96 well plates Life Technologies Cat no AM10027 or AM10050 e 37 C heat block or water bath e Optional MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 Before you begin Ifyou have not done so already add 44 mL of 100 ethanol to the Wash Solution Concentrate and mix well Mark the label on the bottle to indicate that you added
44. annel P200 pipettor at 150 uL Attach new 200 uL tips to the pipettor then mix the suspension in each well thoroughly by pipetting the wells up and down 10 times Note The color of the mixture should be homogeneous after mixing e Incubate the samples for 5 minutes at room temperature off of the magnetic stand 3 Remove the supernatant from the beads a Place the Processing Plate on a magnetic stand for 5 6 minutes to separate the beads from the solution Wait for the solution to clear before proceeding to the next step b Leave the Processing Plate on the magnetic stand then aspirate and discard the supernatant from the plate IMPORTANT Do not disturb the magnetic beads If any beads are aspirated leave 2 3 microliters of supernatant behind 4 Wash the beads with Wash Solution Concentrate with ethanol a Leave the Processing Plate on the magnetic stand b Add 150 uL of Wash Solution Concentrate with ethanol to each sample c Incubate the samples at room temperature for 30 seconds 5 Remove the supernatant from the beads a Aspirate and discard the supernatant from the plate lon Total RNA Seq Kit v2 User Guide 31 Chapter 2 Prepare Whole Transcriptome Libraries Construct the whole transcriptome library b Use a P10 or P20 pipettor to remove residual ethanol IMPORTANT Do not disturb the separated magnetic beads Remove all of the Wash Solution Concentrate from each well c Air dry the beads at room t
45. antify the percentage of DNA that is X160 bp Use size range 50 160 bp b Determine the molar concentration nM of the cDNA libraries Use size range 50 1000 bp Note For instructions on how to perform the smear analysis see Perform a smear analysis on page 68 and refer to the Agilent 2100 Bioanalyzer Expert User s Guide Pub no G2946 90000 4 Use molar concentration of the cDNA libraries from 3b of Pool barcoded whole transcriptome libraries on page 33 and Determine the library dilution required for template preparation on page 34 32 lon Total RNA Seq Kit v2 User Guide Chapter 2 Prepare Whole Transcriptome Libraries Pool barcoded whole transcriptome libraries Next steps If the percent of DNA in 50 160 bp the range is Then 5096 Proceed to Determine the library dilution required for template preparation on page 34 or Pool barcoded whole transcriptome libraries on page 33 50 60 Perform another round of purification on the amplified DNA using components from the Magnetic Bead Cleanup Module 1 Bring the sample volume to 53 uL with Nuclease Free Water 2 Follow the steps in Purify the cDNA on page 27 or Proceed to Determine the library dilution required for template preparation on page 34 or Pool barcoded whole transcriptome libraries but expect to see a slightly higher percentage of filtered reads in your sequencing data when compared to libraries with less than 5
46. cleotides 1000 lon Total RNA Seq Kit v2 User Guide Chapter 2 Prepare Whole Transcriptome Libraries Fragment the whole transcriptome RNA Figure 2 Size distribution of fragmented HeLa rRNA depleted total RNA 500 Fluorescence ro o o 100 25 200 500 1000 2000 4000 Nucleotides lon Total RNA Seq Kit v2 User Guide 23 Chapter 2 Prepare Whole Transcriptome Libraries Construct the whole transcriptome library Construct the whole transcriptome library IMPORTANT The Ion Adaptor Mix v2 Ion RT Primer v2 and Ion PCR primers are unique to the Ion Total RNA Seq Kit v2 Do not use the reagents from the Ion Total RNA Seq Kit first version to prepare libraries with this user guide Constructing the whole transcriptome library involves the following procedures Hybridize and ligate the RNA page 24 y Perform reverse transcription RT page 26 4 Purify the cDNA page 27 i Amplify the cDNA page 29 i Assess the yield and size distribution of the amplified DNA page 32 i Pool barcoded whole transcriptome libraries page 33 i Determine the library dilution required for template preparation page 34 l Proceed to template preparation page 34 Hybridize and Use components from the Ion Total RNA Seq Kit v2 ligate the RNA Ion Adaptor Mix v2 Hybridization Solution e Nuclease Free Water 2XLigation Buffer Ligation Enzyme Mix 1 On ice prepare the hybridization master mix
47. d DNA yields for HeLa poly A RNA and HeLa rRNA depleted total RNA are greater than 200 ng in a 15 uL final volume Workflow Input Amount Typical Recovery Amount Fragment the whole transcriptome 500 ng of poly A RNA total RNA or 300 400 ng of RNA RNA page 17 rRNA depleted total RNA Construct the whole transcriptome lt 1 100 ng of fragmented RNA 5 ng of cDNA library page 24 lon Total RNA Seq Kit v2 User Guide 39 Troubleshooting Troubleshooting Chapter 2 Prepare Whole Transcriptome Libraries Observation Possible Cause Solution The Agilent software does not calculate one concentration and peak size The software detects multiple peaks in the amplified cDNA profile Refer to Analyze multiple peaks as one peak on page 69 Low yield and poor size distribution in the amplified library You recovered lt 20 of the input RNA after you fragmented and cleaned up the RNA Decrease the RNase III digestion from 10 minutes to 5 minutes step 3 on page 19 Low yield in the amplified library and very few differences in the Agilent 2100 Bioanalyzer instrument traces before and after you fragment the RNA RNA fragmentation failed Purify the RNA sample again to remove the extra salts that may affect the RNase lll activity If RNA fragmentation still fails increase the RNase lll digestion from 10 minutes to 20 minutes step 3 on page 19 Low yield and no PCR products
48. e The whole transcriptome RNA library concentration is 400 nM Volume per template preparation reaction is 20 uL lon Total RNA Seq Kit v2 User Guide Chapter 3 Prepare Small RNA Libraries Proceed to template preparation Dilute 1 uL of library in 99 uL of Nuclease Free Water 1 100 dilution to yield a final library concentration of 4 nM Use this final library concentration to calculate the Template Dilution Factor Template Dilution Factor 4 nM x 5 x 109 molecules uL 8 3 nM x 20 uL 210 x 106 molecules 229 Thus 1 uL of the 4 nM library dilution mixed with 228 uL of Nuclease Free Water 1 229 dilution yields approximately 210 x 106 molecules per 20 uL Proceed to template preparation The library is ready for the template preparation procedure In this procedure each library template is clonally amplified on Ion Sphere Particles for sequencing on the Ion PGM System or Ion PI Ion Sphere Particles for sequencing on the Ion Proton System For instructions refer to the specific user guide for an appropriate Ion template preparation kit Template preparation documentation is available on the Ion Community at http ioncommunity iontorrent com Follow the links under Protocols Prepare Template gt Prepare Template User Guides and Quick Reference You may also refer to the Preparation and Sequencing of RNA Libraries with the Ion Personal Genome Machine PGM System User Bulletin Pub no 4478119 T
49. e Technologies Catalog no AM10027 or AM10050 e 37 C heat block or water bath e Optional MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 Before you begin Add 44 mL of 100 ethanol to the bottle of Wash Solution Concentrate and mix well Mark the label on the bottle to indicate that you added ethanol Store the solution at room temperature 15 C to 30 C e If you see a white precipitate in the Binding Solution Concentrate warm the solution at 37 C then shake the solution to dissolve any precipitate before use e Incubate the Nuclease Free Water at 37 C for 25 minutes Note To reduce the chance of cross contamination we strongly recommend sealing unused wells on the Processing Plate with MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 You can also skip a row between sample rows IMPORTANT Accurate pipetting of bead cleanup reagents is critical for best results lon Total RNA Seq Kit v2 User Guide 19 Chapter 2 Prepare Whole Transcriptome Libraries Fragment the whole transcriptome RNA 1 Prepare beads for each sample a Gently vortex the Nucleic Acid Binding Beads tube to completely resuspend the magnetic beads b Add 5 uL beads to wells on the Processing Plate c Add 90 uL Binding Solution Concentrate to each well then mix the Concentrate and beads by pipetting up and down 10 times 2 Bind the fragment RNA products to the beads a Transfer each 30 uL fragment RN
50. e amplified cDNA profile Refer to Analyze multiple peaks as one peak on page 69 Low yield in the desired size range and high background of smaller lt 86 bp non barcoded library or lt 94 bp for barcoded small RNA library or gt 106 bp for non barcoded library or gt 114 bp for barcoded library Ethanol concentration is incorrect during bead size selection 1 Ensure that the ethanol is 100 or 200 proof absolute Sub optimal library size selection with lower ethanol percentage could generate libraries with larger RNA species such as tRNA and 5S 5 8S rRNA 2 Follow the protocol exactly Some of the steps such as pre wetting the tip are critical for accurate pipetting and correct size selection 3 Calibrate your pipettor 4 Using the remaining half of cDNA repeat PCR and PCR cleanup Low yield or only self ligation products are visible on Bioanalyzer instrument traces Your input amount is too low Use enriched or purified small RNA instead of total RNA for ligation or increase the ligation time to 16 hours An enzymatic reaction is not optimal Before eluting cDNA from the Nucleic Acid Binding Beads ensure that the beads are completely dry before adding elution buffer Residual ethanol can inhibit PCR Normal or high yield but PCR products larger than 150 bp Too many PCR cycles resulted in overamplification Decrease the number of PCR cycles step 3 on page 56
51. emperature to remove all traces of ethanol by leaving the Processing Plate on the magnetic stand for 1 2 minutes IMPORTANT Do not overdry the beads overdried beads appear cracked Overdrying significantly decreases elution efficiency 6 Elute the cDNA from the beads a Remove the Processing Plate from the magnetic stand b Add 15 uL of pre warmed 37 C Nuclease Free Water to each sample then mix the Nuclease Free Water and beads by pipetting up and down 10 times c Incubate at room temperature for 1 minute d Place the Processing Plate on a magnetic stand for 1 minute to separate the beads from the solution Wait for the solution to clear before proceeding to the next step e For each sample collect the eluant Assess the yield Use a NanoDrop Spectrophotometer or the dsDNA HS Assay Kit with the Qubit and size Fluorometer Also use the Agilent 2100 Bioanalyzer instrument with the Agilent distribution of the DNA 1000 Kit or Agilent High Sensitivity DNA Kit amplified DNA 1 Measure the concentration of the purified DNA with a NanoDrop Spectrophotometer or the dsDNA HS Assay Kit with the Qubit Fluorometer 2 Analyze 1 uL of the library using the appropriate chip on the Agilent 2100 Bioanalyzer instrument If the library concentration is e 1 50 ng uL Use the Agilent DNA 1000 Kit e 5 1000 pg uL Use the Agilent High Sensitivity DNA Kit 3 Using the 2100 expert software perform a smear analysis to a Qu
52. ent 2100 Bioanalyzer instrument with the Small RNA Kit chip Follow the manufacturer s instructions for performing the assay 2 Compare the bioanalyzer traces to those of the sample before enrichment see step 2 in Assess the amount and quality of small RNA in your total RNA samples on page 43 and determine whether the RNA is degraded For enriched small RNA samples peaks should be from 10 200 nt Using the results from the Agilent 2100 Bioanalyzer instrument and the Small RNA Kit determine the amount of total RNA to use according to the type of RNA you ran and the amount of miRNA in 3 uL If necessary concentrate the small RNA with a SpeedVac centrifugal concentrator Input Sample Type Amount of miRNA 10 40 nt in 3 pL sten Total RNA 5 100 ng lt 1 ug Enriched small RNA 1 100 ng lt 1 ug t The yield drops if you use more than 1 ug of RNA for ligation Note When starting from total RNA with low RIN the miRNA quantity could be over estimated on the Agilent Small RNA chip so more input into ligation is recommended Ideally use gt 10 ng of miRNA lon Total RNA Seq Kit v2 User Guide Chapter 3 Prepare Small RNA Libraries 3 Construct the small RNA library Construct the small RNA library Hybridize and ligate the RNA Constructing the amplified small RNA library involves the following procedures Hybridize and ligate the RNA page 49 i Perform reverse transcription page 51 y Purify and
53. er 3 Prepare Small RNA Libraries Prepare the starting material Add 7 uL beads to the wells of the Processing Plate Set a single or multi channel P200 pipettor at 150 uL Attach new 200 uL tips to the pipettor then mix the suspension in each well thoroughly by pipetting the wells up and down 10 times IMPORTANT Due to the large volume in each well use a P200 pipettor for mixing to avoid cross well contamination Note The color of the mixture should be homogeneous after mixing Incubate the samples for 5 minutes at room temperature off of the magnetic stand 9 Remove the supernatant from the beads a Place the Processing Plate on a magnetic stand for 5 minutes to separate the beads from the solution Wait for the solution to clear before proceeding to the next step Leave the Processing Plate on the magnetic stand then carefully aspirate and discard the supernatant from the plate IMPORTANT Do not disturb the magnetic beads If any beads are aspirated leave 2 3 microliters of supernatant behind 6 Wash the beads with the Wash Solution Concentrate with ethanol a Leave the Processing Plate on the magnetic stand then add 150 uL of Wash Solution Concentrate with ethanol to each sample Incubate the samples for 30 seconds Leave the Processing Plate on the magnetic stand then aspirate and discard the supernatant from the plate IMPORTANT Do not disturb the separated magnetic beads Remove al
54. ess RNA Seq Barcode 01 16 Kit Required for library preparation 12 For the Safety Data Sheet SDS of any chemical not distributed by Life Technologies contact the chemical manufacturer Before handling any chemicals refer to the SDS provided by the manufacturer and observe all relevant precautions Sufficient PCR primers are supplied in the Ion Xpress RNA Seq Barcode 01 16 Kit Cat no 4475485 to prepare barcoded cDNA libraries from 12 samples Components Cap color Quantity Volume Storage lon Xpress RNA BC 01 BC 16 White 1 each 12 uL 30 C to 10 C lon Xpress RNA 3 Barcode Primer Blue 1 each 192 uL Item Source Cat No Thermal cycler with heated lid capable of holding 0 2 mL tubes e Veriti 96 Well Thermal Cycler e GeneAmp PCR System 9700 Life Technologies e 4375786 Various Agilent 2100 Bioanalzyer instrument Agilent G2938A NanoDrop Spectrophotometer Thermo Scientific Qubit 2 0 Fluorometer Life Technologies Q32866 Centrifugal vacuum concentrator for example SpeedVac Major Laboratory Supplier MLS Microcentrifuge MLS Pipettors positive displacement or air displacement MLS Magnetic stand one of the following Life Technologies Magnetic Stand 96 e AM10027 96 well Magnetic Ring Stand e AM10050 Optional Multi channel pipettor MLS Agilent DNA 1000 Kit Agilent 5067 1504 Agilent High Sensitiv
55. f the region until the entire library is included FU 120 100 80 40 Drag the lower region Drag the upper region limit limit towards the left towards the right The software recalculates the median size bp concentration ng uL and molarity nM of the peak region and displays the values in the Peak Table 70 lon Total RNA Seq Kit v2 User Guide Appendix A Supplemental Information Using 2100 expert software to assess whole transcriptome libraries FU 120 100 HeLa PolyA 200 300 400 500 700 1500 bp Conc ng ul ndis Observations Area 4 20 424 2 Lower Marker l 70 5 1 lon Total RNA Seq Kit v2 User Guide 71 Appendix A Supplemental Information Using 2100 expert software to assess small RNA libraries Using 2100 expert software to assess small RNA libraries Review the median The 2100 expert software automatically calculates the median size bp of miRNA size ligation products Select the Peak Table tab then review the median size in the Peak Table and at the top of the peak in the electropherogram The median size should be 87 91 bp FU id enriched placenta RNA 50 40 30 20 15 100 200 300 400 s500 700 1500 bp Name From bp To bp Area of Total Color Conc ngid Molarity nmol T Region 1 50 300 445 99 M 6 97 106 2 2 Region 2 86 106 303 67 MEN 4 76 73 3 Perform a smear Perform a smear analysis to quantify the percentage of DNA in the 5
56. g and with increased ethanol concentration desired cDNA products miRNA and other small RNA in the supernatant re bind to the magnetic beads After washing the beads the desired cDNA products are eluted with pre warmed 37 C Nuclease Free Water Required materials from the Magnetic Bead Cleanup Module Wash Solution Concentrate Binding Solution Concentrate Nucleic Acid Binding Beads Processing Plate e Nuclease Free Water Other materials and equipment 100 ethanol or 200 proof absolute ethanol ACS grade or higher quality Magnetic stand for 96 well plates Life Technologies Cat no AM10027 or AM10050 e 37 C heat block or water bath e Optional MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 Before you begin Ifyou have not done so already add 44 mL of 100 ethanol to the Wash Solution Concentrate and mix well Mark the label on the bottle to indicate that you added ethanol Store the solution at room temperature 15 C to 30 C Ifyou see a white precipitate in the Binding Solution Concentrate warm the solution at 37 C then shake the solution to dissolve any precipitate before use Incubate the Nuclease Free Water at 37 C for 25 minutes Note To reduce the chance of cross contamination we strongly recommend sealing unused wells on the Processing Plate with MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 You can also skip a row between sample rows IMPORT
57. h pre warmed 80 C Nuclease Free Water Required materials from the Magnetic Bead Cleanup Module Wash Solution Concentrate Binding Solution Concentrate Nucleic Acid Binding Beads Processing Plate Nuclease Free Water Note The 1 5 mL Non Stick RNAse free Microfuge Tubes Cat no AM12450 may be used in place of the Processing Plate Other materials and equipment 100 ethanol or 200 proof absolute ethanol ACS grade or higher quality Magnetic rack or stand e 80 C heat block or water bath Optional MicroAmp Clear Adhesive Film Before you begin Add 44 mL of 100 ethanol to the bottle of Wash Solution Concentrate and mix well Mark the label on the bottle to indicate that you added ethanol Store the solution at room temperature 15 C to 30 C e If you see a white precipitate in the Binding Solution Concentrate warm the solution at 37 C then shake the solution to dissolve any precipitate before use Incubate the Nuclease Free Water at 80 C for 25 minutes Note To reduce the chance of cross contamination we strongly recommend sealing unused wells on the Processing Plate with MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 You can also skip a row between sample rows IMPORTANT Accurate pipetting of bead cleanup reagents is critical to successful size selection For optimal size selection perform the following bead cleanup steps exactly 1 Prepare beads for each sample a
58. hanol e Air dry the beads at room temperature for 1 2 minutes to remove all traces of ethanol IMPORTANT Do not overdry the beads overdried beads appear cracked overdrying significantly decreases elution efficiency 7 Elute the cDNA from the beads a Remove the Processing Plate from the magnetic stand b Add 12 uL of pre warmed 37 C Nuclease Free Water to each sample c Mix thoroughly by pipetting up and down 10 times d Incubate the samples at room temperature for 1 minute e Place the Processing Plate on the magnetic stand for 1 minute to separate the beads from the solution Wait for the solution to clear before proceeding to the next step f For each sample collect 12 uL of the eluant Amplify the cDNA To prepare non barcoded libraries use the following components from the Ion Total RNA Seq Kit v2 e on 5 PCR Primer v2 on3 PCR Primer v2 Platinum PCR SuperMix High Fidelity 54 lon Total RNA Seq Kit v2 User Guide Chapter 3 Prepare Small RNA Libraries 3 Construct the small RNA library To prepare barcoded libraries plan the barcodes that you want to use then select the PCR primers from Ion Xpress RNA Seq Barcode 01 16 Kit Ion Xpress RNA Seq Barcode BC 01 BC 16 on Xpress RNA 3 Barcode Primer 1 For each cDNA sample prepare the PCR mix according to the preparation of a non barcoded or barcoded library IMPORTANT Use the appropriate primers Non barcoded Library
59. he Region Table review the percentage of the total product in the size ranges you set From bp To bp Area of Total Color 25 160 266 5 22 MIN Analyze multiple On the Peak Table tab you may observe that the bioanalyzer software identified peaks as one peak multiple peaks in a region that you want to consider as one peak To obtain one concentration and automatically determine the median size for a peak region manually set the size range of the desired peak region 1 In the bottom left corner of the software window select the Peak Table tab Best Peak ral regon Table Legend 2 Right click anywhere on the electropherogram then select Manual Integration FU HeLa PolyA 120 100 40 P B 0 Copy Electropherogram JE Save Electropherogram DS Manual Integration 15 100 bp 3 To remove multiple peaks lon Total RNA Seq Kit v2 User Guide 69 Appendix A Supplemental Information Using 2100 expert software to assess whole transcriptome libraries a Place the cursor on the peak to remove right click then select Remove Peak FU HeLa PolyA 120 100 40 D Copy Electropherogram bp Jp Save Electropherogram FE Manual Integration c Add Peak 15 100 200 Size bp Conc ng pl Molarity nmol l 41 15 4 20 424 2 165 13 61 124 b Repeat until one peak remains within the region of interest c Drag the lower and upper region limits o
60. he bottom of the tube Incubate in a thermal cycler with a heated lid at 70 C for 10 minutes then snap cool on ice 3 Perform the reverse transcription reaction a Add 4 uL of 10X SuperScript III Enzyme Mix to each ligated RNA sample b Gently vortex to mix thoroughly then centrifuge briefly c Incubate in a thermal cycler with a heated lid at 42 C for 30 minutes 26 lon Total RNA Seq Kit v2 User Guide Purify the cDNA Chapter 2 Prepare Whole Transcriptome Libraries Construct the whole transcriptome library STOPPING POINT The cDNA can be stored at 30 C to 10 C for 2 weeks stored at 86 C to 68 C for long term storage or used immediately Required materials from the Magnetic Bead Cleanup Module Wash Solution Concentrate Binding Solution Concentrate Nucleic Acid Binding Beads Processing Plate e Nuclease Free Water Other materials and equipment 100 ethanol or 200 proof absolute ethanol ACS grade or higher quality Magnetic stand for 96 well plates Life Technologies Cat no AM10027 or AM10050 e 37 C heat block or water bath e Optional MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 Before you begin Ifyou have not done so already add 44 mL of 100 ethanol to the Wash Solution Concentrate and mix well Mark the label on the bottle to indicate that you added ethanol Store the solution at room temperature 15 C to 30 C e If you see a white precipitate in the
61. ing Plate on the magnetic stand then add 150 uL of Wash Solution Concentrate with ethanol to each sample Incubate the plate at room temperature for 30 seconds Leave the Processing Plate on the magnetic stand then aspirate and discard the supernatant from the plate Usea P10 or P20 pipettor to remove residual ethanol IMPORTANT Do not disturb the separated magnetic beads Remove all of the Wash Solution Concentrate from each well Air dry the beads at room temperature for 1 2 minutes to remove all traces of ethanol IMPORTANT Do not overdry the beads overdried beads appear cracked overdrying significantly decreases elution efficiency lon Total RNA Seq Kit v2 User Guide Chapter 3 Prepare Small RNA Libraries 3 Construct the small RNA library 7 Elute the cDNA from the beads Remove the Processing Plate from the magnetic stand a b Add 15 uL of pre warmed 37 C Nuclease Free Water to each sample Q Mix thoroughly by pipetting up and down 10 times d Incubate the Processing Plate at room temperature for 1 minute e Place the Processing Plate on the magnetic stand for 1 minute to separate the beads from the solution Wait for the solution to clear before proceeding to the next step f For each sample collect the 15 uL of eluant Assess the yield Use the Agilent 2100 Bioanalyzer instrument with the Agilent DNA 1000 Kit and Size 1 Run 1 uL of the purified DNA on an Agilent 2100 Bioanal
62. ite 1 30 uL 10X SuperScript Ill Enzyme Yellow 1 56 uL Mix Platinum PCR SuperMix Blue 1 900 uL High Fidelity WT Control RNA 1 ug uL Clear 1 50 uL HeLa total RNA Small RNA Control 1 ug uL Purple 1 10 pL human placenta total RNA lon RNA Seq Primer lon Adaptor Mix v2 Green 1 tube 30 uL 30 C to 10 C Set v2 12 Reaction E Kit Part no 4474810 lon RT Primer v2 Yellow 1 tube 104 uL lon 5 PCR Primer v2 White 1 tube 20 uL lon 3 PCR Primer v2 Blue 1 tube 20 uL Magnetic Bead Processing Plate 1 15 C to 30 C TAR porn Binding Solution Concentrate 1 bottle 11 mL room temperature Wash Solution Concentrate 1 bottle 11 mL Nucleic Acid Binding Beads Clear 1 tube 0 7 mL 2 C to 8 C IMPORTANT Do not freeze t Life Technologies has validated this protocol using this specific material Substitution may adversely affect performance 10 lon Total RNA Seq Kit v2 User Guide lon Total RNA Seq Kit v2 48 Reaction Chapter 1 Product Information Kit components and storage Sufficient reagents are supplied in the Ion Total RNA Seq Kit v2 48 Reaction Kit Cat no 4479789 to prepare cDNA libraries from 48 samples for sequencing analysis with the Ion PGM System and Ion Proton System Kit Box Componentst Cap Color Quantity Volume Storage lon RNA Seq Core Kit 10X RNase Ill Reaction Buffer Red 1 60 uL 30 C to 10 C v2 48 Reaction Kit
63. ith higher RIN values 3 Determine the percentage of small RNA in your sample a Run 1 uL of diluted RNA on the Agilent 9 2100 Bioanalyzer instrument with the Small RNA Kit chip Follow the manufacturer s instructions for performing the assay b Using the 2100 expert software determine the mass of total RNA miRNA 10 40 nt from the Small RNA Kit chip c Calculate the miRNA content in your RNA sample using the formula miRNA mass of miRNA mass of total RNA x 100 4 Determine whether small RNA enrichment is needed and the type of enrichment to perform How much miRNA 10 40 nt is in your RNA sample Recommendations for small RNA enrichment and next steps 20 596 miRNA You can use the total RNA in the ligation reaction and small RNA enrichment is not needed However for optimal results we recommend enrichment of all total RNA samples You can expect to see 5 15 more of rRNA and tRNA mapping in your sequencing data from total RNA compared to sequencing data of libraries starting from enriched small RNA Proceed to Enrich the samples for small RNA or skip to Determine the input amount on page 48 lt 0 5 miRNA Small RNA enrichment is strongly recommended We recommend using the Magnetic Bead Cleanup Module for small RNA enrichment Proceed to Enrich the samples for small RNA Note We recommend enriching all total RNA samples for small RNA for optimal results Howeve
64. ity DNA Kit Agilent 5067 4626 Ethanol 100 ACS reagent grade or equivalent MLS Optional Qubit dsDNA HS Assay Kit 100 assays Life Technologies Q32851 8 strip PCR Tubes amp Caps RNase free 0 2 mL Life Technologies AM12230 Non Stick RNase Free Microfuge Tubes 0 5 mL 500 Life Technologies AM12350 Non Stick RNase Free Microfuge Tubes 1 5 mL 250 Life Technologies AM12450 Pipette tips RNase free MLS lon Total RNA Seq Kit v2 User Guide Materials for whole transcriptome libraries Materials for small RNA libraries Chapter 1 Product Information Materials and equipment required but not provided Item Source Cat No Optional 1 2 mL 96 well plates Thermal Fisher AB 1127 Optional FirstChoice Total RNA Life Technologies various cat no Item Source Cat No Qubit RNA Assay Kit 100 assays Life Technologies Q32852 Agilent RNA 6000 Pico Kit Agilent 5067 1513 Optional ERCC RNA Spike In Control Mixes Note ERCC controls are highly recommended Life Technologies 4456739 and 4456740 Optional Dynabeads mRNA DIRECT Micro Kit Life Technologies 61021 Optional MicroPoly A Purist Kit Life Technologies AM1919 Optional TaqMan Gene Expression Assays for ERCC Targets Life Technologies Various cat no Optional TaqMan Gene Expression Master Mix Life Technologies 436901
65. l of the Wash Solution Concentrate from each well Usea P10 or P20 pipettor to remove residual ethanol Air dry the beads at room temperature for 1 2 minutes to remove all traces of ethanol IMPORTANT Do not overdry the beads overdried beads appear cracked overdrying significantly decreases elution efficiency 7 Elute the small RNA from the beads lon Total RNA Seq Kit v2 User Guide Q a Remove the Processing Plate from the magnetic stand b Add 30 uL of pre warmed 80 C Nuclease Free Water to each sample Mix thoroughly by pipetting up and down 10 times Incubate the samples at room temperature for 1 minute Place the Processing Plate on the magnetic stand for 1 minute to separate the beads from the solution Wait for the solution to clear before proceeding to the next step 47 Chapter 3 Prepare Small RNA Libraries Prepare the starting material Assess the quality and quantity of the small RNA enriched sample Determine the input amount 48 f For each sample collect 30 uL of the eluant STOPPING POINT Store the small RNA at 86 C to 68 C After storage proceed to the next section Assess the quality and quantity of the small RNA enriched sample Assess the quality and quantity of samples that are enriched for small RNA Use the Agilent 2100 Bioanalyzer instrument with the Agilent Small RNA Kit 1 Run 1 uL of purified and enriched small RNA sample on the Agil
66. lick the tube or slowly pipet the solution up and down 5 times to mix well then centrifuge briefly to collect the liquid in the bottom of the tube 3 Run the PCRs in a thermal cycler Stage Temp Time Hold 94 C 2 min Cycle 2 cycles 94 C 30 sec 50 C 30 sec 68 C 30 sec Cycle 94 C 30 sec e 16 cycles for 1 5 ng poly A RNA or 62 C 30 sec 10 100 ng rRNA depleted RNA 68 C 30 sec 14 cycles for gt 5 ng poly A RNA or 100 ng rRNA depleted RNA Hold 68 C 5 min Purify the Required materials from the Magnetic Bead Cleanup Module amplified cDNA e Wash Solution Concentrate Binding Solution Concentrate Nucleic Acid Binding Beads Processing Plate Nuclease Free Water Other materials and equipment 100 ethanol or 200 proof absolute ethanol ACS grade or higher quality Magnetic stand for 96 well plates Life Technologies Cat no AM10027 or AM10050 37 C heat block or water bath Optional MicroAmp Clear Adhesive Film Life Technologies Cat no 4306311 Before you begin If you have not done so already add 44 mL of 100 ethanol to the Wash Solution Concentrate and mix well Mark the label on the bottle to indicate that you added ethanol Store the solution at room temperature 15 C to 30 C If you see a white precipitate in the Binding Solution Concentrate warm the solution at 37 C then shake the solution to dissolve any precipitate before use Incubate the Nuclease Free Water at 37 C for 25 minutes Note To reduce
67. lution Factor You can also determine the molar concentration of the pooled libraries with the Agilent DNA 1000 Kit or the Agilent High Sensitivity DNA Kit see Assess the yield and size distribution of the amplified DNA on page 32 and Using 2100 expert software to assess whole transcriptome libraries on page 68 lon Total RNA Seq Kit v2 User Guide 33 Chapter 2 Prepare Whole Transcriptome Libraries Determine the library dilution required for template preparation Determine the library dilution required for template preparation With less than 50 of the amplified DNA in the correct range you can proceed to the template preparation procedure see Proceed to template preparation to prepare templated beads for sequencing on the Ion PGM System or the Ion Proton System For template preparation using an appropriate Ion template preparation kit determine the library dilution that gives 210 x 106 molecules of template per 20 uL 210 x 106 is the recommended library input for Whole Transcriptome libraries Use a conversion factor of 8 3 nM 5 x 10 molecules uL and the following formula Template Dilution Factor Library concentration in nM x 5 x 10 molecules uL 8 3 nM x volume per template preparation reaction inuL 210 x 106 molecules For the volume per template preparation reaction refer to the specific user guide for the appropriate Ion template preparation kit If your library concentration is gt 20
68. nM serially dilute 1 10 or 1 100 until your library concentration is X20 nM This step helps prevent a Template Dilution Factor of several thousands Example The whole transcriptome RNA library concentration is 400 nM Volume per template preparation reaction is 20 uL Dilute 1 uL of library in 99 uL of Nuclease Free Water 1 100 dilution to yield a final library concentration of 4 nM Use this final library concentration to calculate the Template Dilution Factor Template Dilution Factor 4 nM x 5 x 109 molecules uL 8 3 nM x 20 uL 210 x 106 molecules 229 Thus 1 uL of the 4 nM library dilution mixed with 228 uL of Nuclease Free Water 1 229 dilution yields approximately 210 x 106 molecules per 20 uL Proceed to template preparation The library is ready for the template preparation procedure In this procedure each library template is clonally amplified on Ion Sphere Particles for sequencing on the Ion PGM System or Ion PI Ion Sphere Particles for sequencing on the Ion Proton System For instructions refer to the specific user guide for an appropriate Ion template preparation kit Template preparation documentation is available on the Ion Community at http ioncommunity iontorrent com Follow the links under Protocols Prepare Template gt Prepare Template User Guides and Quick Reference You may also refer to the Preparation and Sequencing of RNA Libraries with the Ion Personal Genome Machine PGM
69. namic range lower limit of detection and fold change response of your platform The following table provides guidelines for how much Spike In Control Mix to add to the input RNA for whole transcriptome library preparation For detailed information refer to the ERCC RNA Spike In Control Mixes User Guide Volume of Spike In Mix 1 or Mix 2 dilution Amount of Sample RNA Total RNA Poly A RNA 1 ng 1 uL 1 1000 5 ng 5 uL 1 1000 10 ng 1 uL 1 100 50 ng 5 uL 1 100 100 ng 2 uL 1 1000 1 uL 1 10 500 ng 1 uL 1 100 5 uL 1 10 1000 ng 2 uL 1 100 5000 ng 1 uL 1 10 t ERCC RNA Spike In Mix 1 ExFold Spike In Mix 1 or ExFold Spike In Mix 2 ERCC Analysis Plugin The ERCC Analysis plugin is intended to help with ERCC RNA Spike in Controls It enables you to quickly determine whether or not the ERCC results indicate a problem with library preparation or the PGM run For more information about the ERCC Analysis Plugin refer to the ERCC Analysis Plugin User Bulletin Pub no 4479068 Use components from the Ion Total RNA Seq Kit v2 e Nuclease Free Water e 10X RNase III Reaction Buffer e RNase III 1 Onice assemble a reaction for each RNA sample in a 0 2 mL PCR tube Component add in the order shown M EE 1 RNA sample and Nuclease Free Water 8 10 uL e Poly A RNA 1 500 ng rRNA depleted total RNA 10 500 ng e WT Control RNA 500 ng
70. nd amount 2 200e cece eee eee ee 17 ERCC RNA Spike In Control Mixes 00000 eee eee eee aee 17 Fragment the RNA using RNase lll 02 000 18 Purify the fragmented RNA 2 000 c cece eee eee eee ane 19 Assess the yield and size distribution of the fragmented RNA 0 0 00 005 21 Typical results of fragmentation of whole transcriptome RNA 2 00 22 Construct the whole transcriptome library 00 ccc eect ees 24 Hybridize and ligate the RNA 2 22 nannaa ennenen eraren 24 Perform reverse transcription RT 2 0 0 0 0c ccc cnn enn e 26 Put the cDNA codo oe o ce e rue t E tise E EE 27 Ampy the cDNA 29 ee Riva per Snveber E seiten itu Mes eet vbt At en cot 29 Purify the amplified cDNA ssssssssssssssssss s 30 Assess the yield and size distribution of the amplified DNA 0 005 32 Pool barcoded whole transcriptome libraries oooocococcoccccocnono nor 33 Determine the library dilution required for template preparation 00000000 34 Proceed to template preparation 000 nunn re 34 lon Total RNA Seq Kit v2 User Guide 3 Contents Size distributions and yieldS eraci ys ersan rE ERRESA tenet ee e 35 Typi al size distributions enn perm Pe etd Peedi ate ae 35 Expected yields i e wl ene sist ee ed ete Suan De Sa Bea nee 39 Troubleshooting 2 2305 see veces e ta 40 Using a positive control to
71. ol by pipetting the ethanol up and down 3 times Without changing tips add 35 uL of 100 ethanol to each well IMPORTANT While dispensing the ethanol do not force out the last drops Remove the last drop by touching the drop to the well wall Change the tip and repeat steps 4c 4d for the remaining wells only if the tip touches the wall Accurate pipetting of 10076 ethanol is critical for best results Gently vortex the Nucleic Acid Binding Beads tube to completely resuspend the magnetic beads Add 7 uL beads to the wells of the Processing Plate Seta single or multi channel P200 pipettor at 150 uL Attach new 200 uL tips to the pipettor then mix the suspension in each well thoroughly by pipetting the wells up and down 10 times Note The color of the mixture should be homogeneous after mixing Incubate the samples for 5 minutes at room temperature off of the magnetic stand 9 Remove the supernatant from the beads a Place the Processing Plate on a magnetic stand for 5 minutes to separate the beads from the solution Wait for the solution to clear before proceeding to the next step Leave the Processing Plate on the magnetic stand then carefully aspirate and discard the supernatant from the plate IMPORTANT Do not disturb the magnetic beads If any beads are aspirated leave 2 3 microliters of supernatant behind 6 Wash the beads with Wash Solution Concentrate with ethanol a Leave the Process
72. on refer to the Agilent 2100 Bioanalyzer Expert User s Guide Pub no G2946 9000 If the profile for the fragmented RNA does not meet the typical results see Troubleshooting on page 40 for guidance 3 Proceed according to the amount of fragmented RNA you have in 3 uL 21 Chapter 2 Prepare Whole Transcriptome Libraries Fragment the whole transcriptome RNA Typical results of fragmentation of whole transcriptome RNA 22 100 Fluorescence 80 4 60 404 204 Amount of Fragmented RNA in 3 uL Instructions e 250 ng of poly A RNA e 2100 ng of rRNA depleted total RNA 2100 ng of WT Control RNA on page 24 Store the remaining RNA at 86 C to 68 C Proceed to Construct the whole transcriptome library 50 ng of poly A RNA lt 100 of ng rRNA depleted total RNA 1 Dry all of the RNA completely in a centrifugal vacuum concentrator at low or medium heat lt 40 C this should take 10 20 minutes 2 Resuspend in 3 uL of Nuclease Free Water then proceed to Construct the whole transcriptome library on page 24 The figures in this section show profiles from an Agilent 2100 Bioanalyzer instrument after RNase III fragmentation and cleanup Figure 1 shows results with HeLa poly A RNA Figure 2 shows results with HeLa rRNA depleted total RNA Figure 1 Size distribution of fragmented HeLa poly A RNA Mm E un 2000 4000 25 200 500 Nu
73. quipment for example physical containment devices Individuals should be trained according to applicable regulatory and company institution requirements before working with potentially infectious materials Read and follow the applicable guidelines and or regulatory requirements in the following In the U S U S Department of Health and Human Services guidelines published in Biosafety in Microbiological and Biomedical Laboratories found at www cdc gov biosafety Occupational Safety and Health Standards Bloodborne Pathogens 29 CFR 1910 1030 found at www access gpo gov nara cfr waisidx 01 29cfr1910a 01 html e Your company s institution s Biosafety Program protocols for working with handling potentially infectious materials Additional information about biohazard guidelines is available at www cdc gov In the EU Check local guidelines and legislation on biohazard and biosafety precaution and refer to the best practices published in the World Health Organization WHO Laboratory Biosafety Manual third edition found at www who int csr resources publications biosafety WHO CDS CSR LYO 2004 11 en 77 Appendix B Safety Biological hazard safety 78 lon Total RNA Seq Kit v2 User Guide Documentation and Support Obtaining SDSs Safety Data Sheets SDSs are available from www lifetechnologies com support Note For the SDSs of chemicals not distributed by Life Technologies contact the chemical manufactu
74. r if the tissue or cell lines contain sufficient small RNA to allow efficient library preparation skip to Assess the quality and quantity of the small RNA enriched sample on page 48 Based on their source and the RNA isolation method RNA samples vary widely in small RNA content In some tissues the proportion of small RNA is high enough to allow efficient library preparation from total RNA For example the control RNA provided in this kit is total RNA isolated from placenta Many tissues and most cell lines however contain a much smaller fraction of small RNA If the tissues or cell lines that you are using contain a small fraction of small RNA we recommend enrichment of these RNA samples for small RNA Use an Agilent Small RNA chip to measure the concentration of miRNA 10 40 nts in your total RNA or enriched small RNA sample lon Total RNA Seq Kit v2 User Guide Chapter 3 Prepare Small RNA Libraries Prepare the starting material This protocol uses magnetic beads to enrich for small RNA Use the Magnetic Bead Cleanup Module twice with the same sample to size select the desired small RNA products During the first bead binding magnetic beads capture larger RNA species such as mRNA and rRNA During the second binding and with increased ethanol concentration desired small RNA products miRNA and other small RNA in the supernatant re bind to the magnetic beads After washing the beads the desired small RNA products are eluted wit
75. r 10 uL Ligation Enzyme Mix 2 uL Total volume 12 uL t Include 5 10 excess volume to compensate for pipetting error when preparing the master mix IMPORTANT If the 2X Ligation Buffer contains a white precipitate warm the tube at 37 C for 2 5 minutes or until the precipitate is dissolved 2X Ligation Buffer is very viscous pipet slowly to dispense it accurately b Gently vortex to mix then centrifuge briefly to collect the liquid in the bottom of the tube 6 Add 12 uL of ligation master mix to each 8 uL hybridization reaction for a total of 20 uL per reaction 7 Flick the tube or pipet up and down 5 times to mix then centrifuge briefly 50 lon Total RNA Seq Kit v2 User Guide Chapter 3 Prepare Small RNA Libraries 3 Construct the small RNA library 8 Incubate the 20 uL ligation reactions in a thermal cycler at 16 C for 2 16 hours IMPORTANT If the starting enriched small RNA is lt 5 ng we strongly recommend overnight incubation 16 hours at 16 C For a set of experiments we recommend using the same ligation time for all samples to minimize variation IMPORTANT Set the temperature of the thermal cycler lid to match the block temperature turn OFF the heated lid or leave the thermal cycler open during the incubation Perform reverse Required materials from the lon Total RNA Seq Kit v2 transcription Nuclease Free Water e 10X RT Buffer e 2 mM dNTP Mix on RT Primer v2 e 10X SuperScript I
76. rer Obtaining Certificates of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www lifetechnologies com support and search for the Certificate of Analysis by product lot number which is printed on the box Obtaining support For the latest services and support information for all locations go to www iontorrent com support At the website you can e Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions FAQs Submit a question directly to Technical Support Search for user documents SDSs vector maps and sequences application notes formulations handbooks certificates of analysis citations and other product support documents Obtain information about customer training Download software updates and patches lon Total RNA Seq Kit v2 User Guide 79 Documentation and Support lon contact information lon contact information Website www iontorrent com Ion community ioncommunity iontorrent com Support email ionsupport lifetech com Phone numbers In North America 1 87 SEQUENCE 1 877 378 3623 Outside of North America 1 203 458 8552 Addresses 246 Goose Lane 7000 Shoreline Court Suite 100 Suite 201 Guilford CT 06437 South San Francisco CA 94080 USA USA Limited Product Warranty
77. size select the cDNA page 52 i Amplify the cDNA page 54 i Purify and size select the amplified DNA page 56 i Assess the yield and size distribution of the amplified DNA page 59 i Pool barcoded small RNA libraries page 60 y Determine the library dilution required for template preparation page 60 y Proceed to template preparation page 61 Required materials from the lon Total RNA Seq Kit v2 on Adaptor Mix v2 e Hybridization Solution 2XLigation Buffer Ligation Enzyme Mix lon Total RNA Seq Kit v2 User Guide 49 3 Chapter 3 Prepare Small RNA Libraries Construct the small RNA library 1 On ice prepare the hybridization master mix Component ARE for t ne Reaction Hybridization Solution 3 uL lon Adaptor Mix v2 2 uL Total volume 5 HL t Include 5 10 excess volume to compensate for pipetting error when preparing the master mix 2 Add 5 uL of hybridization master mix to 3 uL of small RNA sample 1 100 ng of miRNA in lt 1 ug of enriched small RNA 3 Flick the tube or pipet up and down 5 times to mix then centrifuge briefly to collect the liquid in the bottom of the tube 4 Run the hybridization reaction in a thermal cycler Temperature Time 65 C 10 min 16 C 5 min 5 Onice prepare the ligation master mix a Combine in a 0 5 mL or 1 5 mL Non Stick RNase Free Microfuge Tube Component ptc al ne Reaction 2X Ligation Buffe
78. ss the yield and size distribution of the amplified DNA page 32 v 3 PCR Primer Pool barcoded whole zu pens libraries page 33 mnn RNA sequence Determine the library dilution required for template preparation page 34 v Prepare templated lon Sphere Particles for sequencing on the lon POM System Refer to the specific user guide for an lon template preparation kit Ay A AS P1 B 16 lon Total RNA Seq Kit v2 User Guide Chapter 2 Prepare Whole Transcriptome Libraries Fragment the whole transcriptome RNA Fragment the whole transcriptome RNA Guidelines for RNA sample type and amount ERCC RNA Spike In Control Mixes Fragmenting the whole transcriptome RNA involves the following procedures Fragment the RNA using RNase III page 18 i Purify the fragmented RNA page 19 i Assess the yield and size distribution of the fragmented RNA page 21 We strongly recommend using 1 500 ng of poly A RNA or 10 500 ng of rRNA depleted total RNA You may also use high quality total RNA Guidelines for using poly A RNA To prepare poly A RNA from 100ng 50 ug total RNA we recommend using the Dynabeads mRNA DIRECT Micro Kit Cat no 61021 Refer to the Dynabeads mRNA DIRECT Micro Kit User Guide e 50 400 ug total RNA we recommend performing two rounds of oligo dT selection of the poly A RNA using the MicroPoly A Purist Kit Cat no AM1919 Also confirm the absence of 185 and 285 rRNA for ex
79. sturb the separated magnetic beads Remove all of the Wash Solution Concentrate from each well Air dry the beads at room temperature to remove all traces of ethanol by leaving the Processing Plate on the magnetic stand for 1 2 minutes IMPORTANT Do not overdry the beads overdried beads appear cracked Overdrying significantly decreases elution efficiency 6 Elute the cDNA from the beads Remove the Processing Plate from the magnetic stand Add 12 uL of pre warmed 37 C Nuclease Free Water to each sample then mix the Nuclease Free Water and beads by pipetting up and down 10 times Incubate at room temperature for 1 minute Place the Processing Plate on the magnetic stand for 1 minute to separate the beads from the solution Wait for the solution to clear before proceeding to the next step lon Total RNA Seq Kit v2 User Guide Chapter 2 Prepare Whole Transcriptome Libraries Construct the whole transcriptome library e For each sample collect the eluant Amplify the cDNA To prepare non barcoded libraries use the following components from the Ion Total RNA Seq Kit v2 e on 5 PCR Primer v2 e Ton 3 PCR Primer v2 Platinum PCR SuperMix High Fidelity To prepare barcoded libraries plan the barcodes that you want to use then select the PCR primers from Ion Xpress RNA Seq Barcode 01 16 Kit e on Xpress RNA Seq Barcode BC 01 BC 16 on Xpress RNA 3 Barcode Primer 1 For each cDNA sample
80. t RNA Assay Kit with the Qubit Fluorometer and the Agilent RNA 6000 Pico Kit with the Agilent 2100 Bioanalyzer instrument Note You can use a NanoDrop Spectrophotometer in place of the Qubit RNA Assay Kit and Qubit Fluorometer For increased accuracy quantitate the RNA concentration using the Qubit RNA Assay Kit with the Qubit Fluorometer Note Assessing the yield and size is not recommended for poly A fragmented RNA samples from 5 ng poly A RNA due to low input amount 1 Quantitate the yield of the fragmented RNA using the Qubit RNA Assay Kit with the Qubit Fluorometer Refer to the Qubit RNA Assay Kit Protocol Pub no MAN0002327 or the Qubit Fluorometer Instruction Manual Pub no MAN0002328 for instructions 2 Assess the size distribution of the fragmented RNA a lon Total RNA Seq Kit v2 User Guide If necessary dilute 1 uL of the sample to 50 5000 pg uL with Nuclease Free Water Run the diluted sample on an Agilent 2100 Bioanalyzer instrument with the RNA 6000 Pico Kit Follow the manufacturer s instructions for performing the assay Using the 2100 expert software review the size distribution The fragmentation procedure should produce a distribution of RNA fragment sizes from 35 nt to several hundred or a few thousand nt depending on your sample type The average size should be 100 200 nt See the figures starting on page 22 Note For instructions on how to review the size distributi
81. t or device read and understand the safety information provided in the user documentation provided by the manufacturer of the instrument or device Before handling chemicals read and understand all applicable Safety Data Sheets SDSs and use appropriate personal protective equipment gloves gowns eye protection etc To obtain SDSs see the Documentation and Support section in this document lon Total RNA Seq Kit v2 User Guide 75 Appendix B Safety Chemical safety Chemical safety 76 N WARNING GENERAL CHEMICAL HANDLING To minimize hazards ensure laboratory personnel read and practice the general safety guidelines for chemical usage storage and waste provided below and consult the relevant SDS for specific precautions and instructions Read and understand the Safety Data Sheets SDSs provided by the chemical manufacturer before you store handle or work with any chemicals or hazardous materials To obtain SDSs see the Documentation and Support section in this document Minimize contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves or protective clothing Minimize the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation for example fume hood Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s procedures as recommended in the SDS Handle
82. tes of Analysis 00 cece cece en 79 Obtaining Support inte ses Sa ee EM E geo ARCA REN ERES 79 lori contact information siecle eril eee teddies aee de Due Pee Pa d ds 80 Limited Product Warranty uis sere yen a eus 80 lon Total RNA Seq Kit v2 User Guide 5 Contents 6 lon Total RNA Seq Kit v2 User Guide About This Guide B Revisi n history ione Ee CREUSE ES e RR ace edes 7 m Other Ion library preparation kits and guidesS oooooccccccccccccc 7 m Ambion products and expertise nunes cece eee 7 IMPORTANT Before using this product read and understand the information in Appendix B Safety on page 75 in this document Revision history Revision Date Description of change D 17 December 2012 Combined the whole transcriptome protocol for standard and low RNA inputs Added support for the lon Proton System Placed optional materials in Chapter 1 Product Information and removed index 22 August 2012 Added the new 48 reaction kit the lon Total RNA Seq Kit v2 48 Reaction Kit Cat no 4479789 Other lon library preparation kits and guides Other library preparation kits and protocols are available For guides and protocols visit the Ion community at http ioncommunity iontorrent com and follow the links under Protocols Construct Library Construct Library User Guides and Quick Reference Ambion products and expertise
83. the last drops Remove the last drop by touching the drop to the well wall Change the tip and repeat steps 2c 2d for the remaining wells only if the tip touches the wall Accurate pipetting of 10076 ethanol is critical for best results Set a single or multi channel pipettor at 150 uL Attach new 200 uL tips to the pipettor then mix the suspension in each well thoroughly by pipetting the wells up and down 10 times Note The color of the mixture should be homogeneous after mixing Incubate the samples for 5 minutes at room temperature off of the magnetic stand 3 Remove the supernatant from the beads a Place the Processing Plate on the magnetic stand for 5 6 minutes to separate the beads from the solution Wait for the solution to clear before proceeding to the next step Leave the Processing Plate on the magnetic stand then aspirate and discard the supernatant from the plate IMPORTANT Do not disturb the magnetic beads If any beads are aspirated leave 2 3 microliters of supernatant behind 4 Wash the beads with Wash Solution Concentrate with ethanol a Leave the Processing Plate on the magnetic stand b Add 150 uL of Wash Solution Concentrate with ethanol to each sample c Incubate the samples at room temperature for 30 seconds 5 Remove the supernatant from the beads a Aspirate and discard the supernatant from the plate b Use a P10 or P20 pipettor to remove residual ethanol IMPORTANT Do not di
84. y dilution required for template preparation on page 60 or Pool barcoded small RNA libraries but expect to see an increase in the number of filtered reads no insert tRNA or rRNA mapped reads when compared to samples with greater than 50 ratio of desired miRNA ligation products to overall products lon Total RNA Seq Kit v2 User Guide 59 Chapter 3 Prepare Small RNA Libraries Pool barcoded small RNA libraries Note Samples that are run ona Bioanalyzer instrument typically show 5 8 bp larger than their actual size Pool barcoded small RNA libraries Note If you are not pooling libraries skip this section and proceed to Determine the library dilution required for template preparation on page 60 1 Determine the molar concentration nM of each of the barcoded cDNA libraries with the Agilent DNA 1000 Kit or the Agilent High Sensitivity DNA Kit Note 50 300 bp size range is typically used to determine the library concentration If necessary adjust the range to include all of the library peaks 2 Dilute each barcoded cDNA library to the same molar concentration nM For example if you have 3 different barcoded libraries that are 45 55 65 nM choose a concentration that is equal to or lower than the lowest concentration of the three libraries such as 30 nM Dilute all or part of the library to 30 nM 3 Mix an equal volume of each diluted library to prepare a pool of the barcoded libraries 4 The
85. ypical size distribution Plotted size distributions Review the plotted and tabulated size distributions in the following sections Figure 8 shows the size distribution of non barcoded small RNA library from enriched placenta Figure 9 on page 63 illustrates a typical size distribution of placenta total RNA library Agilent 2100 Bioanalyzer instrument profile For the highest quality libraries the ratio of 86 106 bp DNA 50 300 bp DNA is greater than 50 Figure 10 on page 64 illustrates the size distribution of a barcoded small RNA library prepared from placenta total RNA lon Total RNA Seq Kit v2 User Guide 61 Chapter 3 Prepare Small RNA Libraries Typical size distribution Figure 8 Molar concentration and size distribution of non barcoded library prepared from enriched placenta small RNA Fu 3 13 enriched placenta RNA 40 20 10 HEBES 15 100 200 300 400 S00 700 1500 bp 68 of the total Name From bp To bp _ Area of Total Color Conc ngii Molarity ng The library area under the y Region 1 SO 300 o 6 97 ae concentration curve is 86 106 bp 2 Regon2 86 106 67 ME 4 76 is 106 2 nM Note The amount of tRNA in the final library reflected by the height of the peak that is about 108 bp on the bioanalyzer trace varies depending on the lot of placenta you use Expect to see differences in the ratio of 86 106 bp DNA 50 300 bp DNA when different lots of placenta control RNA are used 62 lon Total RNA Seq
86. yzer instrument with distribution of the the Agilent DNA 1000 Kit Follow the manufacturer s instructions for amplified DNA performing the assay 2 Using the 2100 expert software perform a smear analysis to determine size distribution of the amplified DNA a Measure the area for the DNA that is e 50 300 bp the size range for all of the ligation products e 86 106 bp for non barcoded libraries or 94 114 bp for barcoded libraries the size range for the desired miRNA ligation products b Calculate the ratio of mRNA ligation products in total ligation products using the formula for e Non barcoded libraries Area 86 106 bp Area 50 300 bp e Barcoded libraries Area 94 114 bp Area 50 300 bp c Determine the molar concentration of cDNA libraries using size range 50 300 bp Use this concentration for Pool barcoded small RNA libraries and Determine the library dilution required for template preparation on page 60 Note Adjust the size range to include all library peaks if necessary Note For instructions on how to perform the smear analysis see Perform a smear analysis on page 68 and refer to the Agilent 2100 Bioanalyzer Expert User s Guide Pub no G2946 90000 Next steps If the ratio is Then 250 Proceed to Determine the library dilution required for template preparation or Pool barcoded small RNA libraries on page 60 lt 50 Proceed to Determine the librar
87. ze ranges you set Mame From bp To bp Area of Total Color Conc ng ul Molarity nmol l gt Region 1 50 300 44 5 oo M 6 97 106 2 2 Region2 86 106 30 3 67 MEN 4 76 E lon Total RNA Seq Kit v2 User Guide 73 Appendix A Supplemental Information Using 2100 expert software to assess small RNA libraries Determine the Using the area values from the Region Table calculate the miRNA library in the miRNA library 86 106 bp region as a fraction of the 50 300 bp region using the formula miRNA library Area from 86 106 bp Area from 50 300 bp x 100 Example miRNA library calculation In the example below the miRNA library is 68 miRNA library 30 3 44 5 x 100 68 Fu ili enriched placenta RNA 50 40 20 10 400 S00 700 1500 bp Name From bp To bp Area of Total Color Conc ng l Molarity nmol T gt Region 1 so 300 445 99 MIS 6 97 106 2 2 Region2 86 106 303 7 4 76 73 3 74 lon Total RNA Seq Kit v2 User Guide Chemical safety alli at hea CNOA tee bean ERE 76 Biological hazard safety 2 6 ccc eee eee eens 77 WARNING GENERAL SAFETY Using this product in a manner not specified in the user documentation may result in personal injury or damage to the instrument or device Ensure that anyone using this product has received instructions in general safety practices for laboratories and the safety information provided in this document Before using an instrumen

Ion Total RNA-Seq Kit v2 User Guide (Pub. no. 4476286 Rev. D)

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