Archer™ Universal RNA Fusion Detection v1 for
Contents
1. 9 SIBD US Secold PUB oe ennt Omm EMI n ee DM 10 Step 8 Quantitate Library and Sequence eee nene te tenet tn tete tette taria tn into setatis enin renamen 12 For more information please visit http www enzymatics com archer esee 14 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER by enzymatics Product Description Gene fusions represent an important class of genomic rearrangements in translational research The Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform assay utilizes the power of next generation sequencing to improve the detection of genomic rearrangements over traditional methods such as immunohistochemistry IHC and fluorescence in situ hybridization FISH Modular Assay Format The Archer Universal RNA Fusion Detection v1 for Illumina amp Platform used in conjunction with Archer Assays and MBC Adapters allows users to construct Illumina amp MiSeq ready libraries from total nucleic acid or RNA samples Universal Pu Kit FusionPlex Assays MBC Adapters For Research Use Only Not for use in diagnostic procedures al e es o o o o o Workflow Overview 2 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER gt by enzymatics 20 uL of THA RNA H R2. termed an index Archer Universal RNA Fusion Detection v1 for Illumina Platform utilize 2 indices in combination to distinguish between samples The first index is added just before Step 5 Adapter Ligation and is embedded in the MiSeq Index 2 Barcode Adapters The second index is added in Step 7 Second PCR and is embedded in MiSeq Index 1 Primers within the Second PCR reaction In order to maintain appropriate coverage depth it is recommended to cap each MiSeq run at 30 48 samples per lane In general larger panels with more targets will require higher sequencing coverage depth and should be run with fewer samples per sample See example below Archer Panel of Amplicons Assay Recommended of Samples Lane FusionPlex ALK RET ROS1 Panel 29 30 40 v2 4 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER gt by ue Ps Barcode Diversity The Illumina amp MiSeq will work best when index diversity within a run is high For example if eight samples are included in a run and the user chooses to use only one MiSeq Index 2 Barcode Adapter paired with eight different MiSeq Index 1 Primers the run may fail due to low barcode diversity In this example it is best to use eight different MiSeq Index 2 Barcode Adapters paired with eight different MiSeq Index 1 Primers Input Nucleic Acid Concentration and Purification e Total nucleic acid is the preferred input for this assay
3. aY V gt A EP A Remove the yellow 8 tube strip Each tube in the strip provides a single reaction Centrifuge briefly to ensure lyophilized material is in the bottom of the tube If you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C To the Second Strand cDNA Synthesis tube on ice add Ultra Pure Water SA0021 20 uL First Strand cDNA Synthesis reaction Step 2 7 20 uL Total 40 uL Mix well by pipetting gently up and down 6 8 times Spin briefly to collect contents at the bottom of the tube Incubate at 16 C for 1 hour If a thermal cycler is used for the incubation do not use a heated lid or close the heated lid Do not allow the temperature to rise above 16 C Stopping point Itis OK to stop and store the library at 20 C Step 4 End Repair dA Tailing 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 7 Gently open the End Repair dA Tailing SA0004 foil pouch by tearing along the indents located at the top of the silver package Remove the blue 8 tube strip Each tube in the strip provides a single reaction Centrifuge briefly to ensure lyophilized material is in the bottom of the tube If you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new
4. bead solution back on magnet for 2 minutes 5 16 Carefully transfer 22 uL of the purified library solution to a fresh 200 uL PCR tube or proceed directly to Step 6 Be sure to avoid transferring beads to the fresh tube Stopping point Itis OK to stop and store the library at 20 C Step 6 First PCR NOTE The Archer Universal RNA Fusion Detection v1 for Illumina amp Platform Kit do not contain gene specific primers GSPs in the reaction pellet 6 1 Gently open the First PCR SA0009 foil pouch by tearing along the indents located at the top of the silver package 6 2 Remove the clear 8 tube strip Each tube in the strip provides a single reaction 6 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 6 4 If you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch at 4 C 6 5 Tothe First PCR tube on ice add Purified library DNA Step 5 16 18 uL Liquid GSP1 Mix 2 uL 9 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER i idl E y nizyiTiatiics a Pd P Total 20 uL 6 6 Allow the pellet to dissolve and then pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 6 7 Incubate the reaction as follows Note the ramp rate between 98 C and 68 C con
5. razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C Transfer 40 uL of the Second Strand cDNA Synthesis reaction Step 3 7 into tube containing lyophilized End Repair dA Tailing SA0004 reagents and mix well by pipetting up and down 6 8 times Spin briefly to collect contents at the bottom of the tube Incubate the reaction in a thermal cycler with a heated lid set to gt 100 C and incubate as follows Incubation Incubation Step Temperature Time 1 12 C 15 min 2 37 C 15 min 3 729 15 min 4 4 C Hold Ensure the reaction cools to 4 C and briefly centrifuge End Repair reaction before proceeding Gently open a pouch of Archer MBC Adapters for Illumina or MiSeq Index 2 Barcode Adapters by tearing along the indents located at the top of the silver package Remove the clear 8 tube strip from the foil pouch Each tube in the strip provides a single reaction and each tube contains a different Illumina amp MBC Adapter or MiSeq Index 2 Archer Universal RNA Fusion Detection v1 for Illumina amp Platform IFU AK0024 8 Rev A ARCHER by enzymatics Barcode Adapter For example reactions 1 through 8 correspond to MBC Adapters 1 through 8 or corresponds to Index 2 Barcode Adapters 1 through 8 4 9 1 CRITICAL Upon removing the 8 tube strip from the pouch position the tubes with the hinges to the back and use a permanent marker to label the tubes 1 through 8 from le
6. 0 uL of the End Repaired dA tailed DNA with the annealed Illumina MBC Adapters Step 4 13 into the tube containing Adapter Ligation mix Allow pellet to dissolve and then pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 8 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER Vv enzymatics Ps 5 6 Incubate the reaction as follows If a thermal cycler is used either set the thermal cycler lid to Off or leave it open during the incubation Incubation Incubation Step Temperature Time 1 16 C 30 min 2 22 C 30 min Post Ligation AMPure XP Beads Purification 5 7 Refer to manufacturer s protocol for details on methods of purification 5 8 Add 40 uL of AMPure XP beads to the 50 uL reaction for a ratio of 0 8X 5 9 Vortex well or pipette 10 times to mix and incubate for 5 minutes at room temperature 5 10 Collect beads with magnet for 2 4 minutes or until solution is clear 5 11 Carefully pipette off and discard supernatant without disturbing the beads 5 12 Wash twice with 200 uL of 70 ethanol while on the magnet Spin down and carefully remove remaining supernatant while taking care to not resuspend beads 5 13 After the second wash dry beads at room temperature for 5 minutes 5 14 Elute cDNA in 24 uL of 10 mM Tris HCl Remove tubes from the magnet and thoroughly resuspend the beads with the 10 mM Tris HCl 5 15 Place cDNA
7. Instructions for Use ARCHER by enzymatics Archer Universal RNA Fusion Detection v1 for Illlumina Platform AK0024 8 Table of Contents Archer Universal RNA Fusion Detection v1 for Illumina Platform 1 T Or idum aoeetee 1 Prodguet Deg e100 at ans en a ues IU ERrEI IE INE TIENE 2 Modular Assay TOTMAN m 2 IV EAT OC MN EE E E 2 KEON e H 3 Materials Reguired But NOUSUDDIIGO caraang aE AE AEA 3 Genera Prec MN 0 IN siae a NEEE DE EE A a Ea ELE EEEa SAELE EENEN aA 4 OP O c O QA 4 Sample Mukti pleini ereraa ee ne ETEA TASE ARSA 4 barcode Diversi Yasana ea EE A gerd E 5 Input Nucleic Acid Concentration and Purification cscssescessssssesssesssssscscsssesscscsssessssesssesesecsseesesacecssessenaceseneces 5 Bae YOUU B a E E EE A EEE EEE E EN 5 Doon Or a EE 5 Step T1 Random P an E A rr ret 5 Step 2 First Strand CONA Synthesis asirasi aaneen ar ANAE AE EAA AEEA EENEN Eia 6 Step 3 Second Strand cDNA Synthesis eese eene ne te ntn tenete tnnt ten tote teta tento tete tatnen 6 Sep 4 End Repair dA TINNE coectetuer sun mim ere eu tanec se HU SEHE ESI IURI EIE EROR irt Ci CR E CUEUGE 6 Sep a AN 20 er tot iim 8 Oe e dc
8. M stock vortex briefly to mix 8 3 2 5 Mix 750 uL Hyb Buffer 250 uL 40 pM library in new 1 5 ml microcentrifuge tube and vortex briefly 10 pM Library stock 8 3 2 6 Mix 900 uL 10pM library stock 100 uL 10 pM denatured PhiX and vortex briefly 8 3 2 7 This creates the final loading pool of 10 pM 10 PhiX 8 3 2 8 Add the entire 1000 uL to the MiSeq cartridge and start the run 8 4 Upon completion of the run the data should be analyzed using the Archer Analysis Pipeline http archer enzymatics com 13 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER gt by enzymatics Limitations of Use For Research Use Only Not for use in diagnostic procedures This product was developed manufactured and sold for in vitro use only The product is not suitable for administration to humans or animals SDS sheets relevant to this product are available upon request Illumina is a registered trademark of Illumina Inc For more information please visit http www enzymatics com archer Enzymatics Inc Zym cj I CS amp 100 Cummings Center Suite 407 Beverly MA 01915 mM Phone 888 927 7027 oe e B 14 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A
9. V enzymatics ZA 9 If nucleic acid is from FFPE tissue it is recommended to use Agencourt FormaPure Cat A33342 for extraction General Precautions e Read the entire protocol before beginning e Take note of stopping points where samples can be frozen at 20 C and plan your workflow accordingly e Use good laboratory practices to minimize cross contamination of nucleic acid products e Always use PCR tubes microfuge tubes and pipette tips that are certified sterile DNase and RNase free e Before starting wipe down work area and pipettes with an RNase and DNA cleaning product such as RNase Away Molecular BioProducts Inc San Diego CA e For consistent library amplification ensure the thermal cycler used in this protocol is in good working order and has been calibrated to within the manufacturer s specifications Storage All components of Archer Universal RNA Fusion Detection v1 for Illumina amp Platform Part f AK0024 8 should be stored at 4 C Allow pouches to warm to room temperature before opening sample Multiplexing In order to efficiently utilize the throughput of the MiSeq multiple samples should be sequenced simultaneously Samples can be identified through a unique nucleotide sequence that is part of the adapter attached to the nucleic acid molecule in a given sample during library construction and which is subsequently read during the sequencing process The unique nucleotide sequence is often
10. andom Priming e e C o o c First Strand cDNA Synthesis e 5 o End Repair dA Tailing 6 5 5 9 8 5 Adapter Ligation n a a TT OQ uL Transfer 20 ul Transfer 20 uL H 40 uL Transfer 10 uL H 0 Ws KY he Vena an z ul Transfer 20 uL AMPure XP Purlficatean First PCR 30 T i H Ea 70 ul E 35 3n il puer ALL uantitate Library and Sequence Kit Contents 1 500 mM Tris HCl pH 8 0 SA0020 2 Ultra Pure Water SA0021 3 Ultra Pure Water for Ethanol Dilution SA0022 4 Lyophilized Reagents Step 1 Random Priming SA0001 Step 2 First Strand cDNA Synthesis SA0002 Step 3 Second Strand cDNA Synthesis SA0003 Step 4 End repair dA tailing SA0004 Step 5 Adapter Ligation SA0005 Step 6 First PCR SA0009 Step 7 Second PCR Reactions 1 through 8 SA0013 m mapon Materials Required But Not Supplied 3 GO D QNCMI m O9 a Archer MBC Adapters for Illumina amp Archer MiSeq Index 2 Barcode Adapters 1 thru 8 Archer FusionPlex Assay Cat AK0028 8 Agencourt AMPure XP Beads Cat A63881 Life Technologies DynaMag Cat 12331D 100 ethanol ACS grade KAPA Biosystems Library Quantification Kit Illumina Universal Cat KK4824 Custom Primer Panels designed at http assay enzymatics com Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER i k APR D
11. bes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C Be sure to track which barcodes were used to ensure barcode compatibility when used in later experiments To the Second PCR tube on ice add 754 Purified library DNA Step 6 17 18 uL Liquid GSP2 Mix 2 uL Total 20 uL Allow the pellet to dissolve and then pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube Incubate the reaction as follows Note the ramp rate between 98 C and 68 C consult your instrument user s manual to confirm that this setting is correct Ensure the lid temperature tracks 5 C above the incubation temperature or set the lid to gt 100 C Incubation Temperature Incubation Time of cycles 98 C 30 sec 1 98 C 10 sec 68 C ramp rate of 30 sec 24 2 3 C sec 72 C 3 min 1 4 C HOLD 1 NOTE The number of unique molecules will be reduced when the PCR cycles are increased and can be decreased based on user experience with different amount of input material and specific sample types 11 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER Vv enzymatics Ps Post Second PCR AMPure XP Beads Purification 7 8 7 9 7 10 7 11 7 12 7 13 7 14 7 15 7 16 7 17 Refer to manufacturer s protocol for details on methods of purification Add 16 uL
12. ck on magnet for 2 minutes 6 17 Carefully transfer 22 uL of the purified library solution to a fresh 200 uL PCR tube or proceed directly to Step 7 Be sure to avoid transferring beads to the fresh tube Stopping point Itis OK to stop and store the library at 209C Step 7 Second PCR NOTE The Archer Universal RNA Fusion Detection v1 for Illumina amp 9 Platform do not contain gene specific primers GSPs in the reaction pellet 10 Archer Universal RNA Fusion Detection v1 for Illumina Platform IFU AK0024 8 Rev A ARCHER by enzymatics Lak 325 Tsd 7 4 7 5 7 6 7 7 Gently open the Second PCR SA0013 foil pouch by tearing along the indents located at the top of the silver package Remove the clear 8 tube strip from the foil pouch Each tube in the strip provides a single reaction and each tube contains a different MiSeq Index 1 Barcode Primer 1 through 8 Reactions 1 through 8 correspond to MiSeq Index 1 through 8 7 2 1 CRITICAL Upon removing the 8 tube strip from the pouch position the tubes with the hinges to the back and use a permanent marker to label the tubes 1 through 8 from left to right as shown below Be sure the label is placed where it will not be compromised when placed in a thermal cycler Centrifuge briefly to ensure lyophilized material is in the bottom of the tube If you would like to use fewer than eight reactions detach the appropriate number of tu
13. e DONOT treat the extracted total nucleic acid with DNase as this will critically reduce the quality of RNA in the sample e Ifnucleic acid is from FFPE tissue it is recommended to use Agencourt FormaPure 9 A33342 for extraction e When possible it is recommended to increase the total nucleic acid input which will increase library complexity and improve the sensitivity of the assay If higher library complexity is desired the assay can tolerate up to 250 ng of total nucleic acid e The minimum recommended input for the assay is 20 ng of total nucleic acid Alternatively 10 ng of RNA may be used e Efficient library preparation can be achieved with as little as 2 ng of total nucleic acid provided that the starting material is of high quality and is not degraded However reduced input will decrease library complexity due to the restricted amount of starting unique target molecules When using less than 10 ng of input material the PCR cycling conditions Steps 6 and 7 may need to be altered e Theuse of EDTA containing buffers in this protocol may result in lower library yields Be sure to use buffers that do not contain EDTA i e use Tris HCl and not Tris EDTA buffer Before You Begin e Make fresh 10 mM Tris HCl o Mix20 uL 500 mM Tris HCl pH 8 0 SA0020 with 980 uL Ultra Pure Water SA0021 e Make fresh 70 ethanol o Add 14 mL 100 ethanol ACS grade not included to entire bottle containing Ultra Pure Water for Ethanol Diluti
14. ft to right as shown below Be sure to label and track the index number added to each sample from this point forward 4 10 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 4 11 If you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C Be sure to track which indices were used to ensure index compatibility when used in later experiments 4 12 To the Illumina MBC Adapters tube or MiSeq Index 2 Barcode Adapters on ice add Ultra Pure Water SA0021 10 uL Total 50 uL 4 13 Allow the pellet to dissolve and then pipet up and down 6 8 times to mix Spin briefly to collect contents at the bottom of the tube 4 14 Immediately proceed to Step 5 Step 5 Adapter Ligation 5 1 Gently open the Adapter Ligation 5A0005 foil pouch by tearing along the indents located at the top of the silver package 5 2 Remove the red 8 tube strip Each tube in the strip provides a single reaction 5 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 5 4 If you would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C 5 5 Transfer 5
15. of AMPure XP beads to the reaction for a ratio of 0 8X Vortex well or pipette 10 times to mix and incubate for 5 minutes at room temperature Collect beads with magnet for 2 4 minutes or until solution is clear Carefully pipette off and discard supernatant without disturbing the beads Wash twice with 200 uL of 70 ethanol while on the magnet After the second wash dry beads at room temperature for 5 minutes Elute cDNA in 24 uL of 10 mM Tris HCl Remove tubes from the magnet and thoroughly resuspend the beads with the 10 mM Tris HCl Place cDNA bead solution back on magnet for 2 minutes Carefully transfer 24 uL of the purified cDNA solution to a fresh 200 uL PCR tube or proceed directly to Step 8 Be sure to avoid transferring beads to the fresh tube Stopping point It is OK to stop and store the library at 20 C Step 8 Quantitate Library and Sequence 8 1 8 2 8 3 12 Use the KAPA Biosystems qPCR kit KK4824 for Illumina to quantitate the concentration of each library Assume a 250 bp fragment length After quantification pool the barcoded libraries at equimolar concentrations and sequence on an Illumina MiSeq It is recommended to cap each MiSeq run at 48 samples to maintain appropriate coverage depth An equimolar library pooling 2 nM or 4 nM pool calculation spreadsheet is available for download at http www enzymatics com archer Run the MiSeq using the read level sequence in the table below In addi
16. on SA0022 o Note the date on which ethanol is added 70 ethanol is appropriate for use for one week after mixing When not in use tightly close the bottle cap to ensure minimal evaporation Instructions for Use Step 1 Random Priming 1 1 Pre heat thermal cycler to 65 C with a heated lid 1 2 Gently open the Random Priming SA0001 foil pouch by tearing along the indents located at the top of the silver package 1 3 Remove the green 8 tube strip Each tube in the strip provides a single reaction 5 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A PH WwW gt ARCHER ENS enzymatics m ni dmm o K 1 4 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 1 5 Ifyou would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with desiccant provided at 4 C 1 6 Place the tubes on ice and to each add Ultra Pure Water SA0021 20 X wh Purified Total Nucleic Acid X uL Total 20 uL 1 7 After the lyophilized pellet dissolves gently pipet up and down 6 8 times and briefly spin down 1 8 Transfer the tubes from ice to the thermal cycler and incubate at 65 C for 5 minutes 1 9 Remove tubes from thermal cycler and place on ice for 2 minutes then briefly centrifuge before proceeding with First Strand cDNA Syn
17. sult your instrument user s manual to confirm that this setting is correct Ensure the lid temperature tracks 5 C above the incubation temperature or set the lid to 2100 C Incubation Incubation Temperature Time of cycles 98 C 30 sec 1 98 C 10 sec 20 68 C ramp rate of 2 3 C sec 30 sec 72 6 3 min 1 4 C HOLD 1 NOTE If library yields are too low the cycle number can be increased up to 22 cycles The number of unique molecules will be reduced when the PCR cycles are increased and can be decreased based on user experience with different amount of input material and specific sample types Post First PCR AMPure XP Beads Purification 6 8 Refer to manufacturer s protocol for details on methods of purification 6 9 Add 16 uL of AMPure XP beads to the 20 uL reaction for a ratio of 0 8X 6 10 Vortex well or pipette 10 times to mix and incubate for 5 minutes at room temperature 6 11 Collect beads with magnet for 2 4 minutes or until solution is clear 6 12 Carefully pipette off and discard supernatant without disturbing the beads 6 13 Wash twice with 200 uL of 70 ethanol while on the magnet Spin down and carefully remove remaining supernatant while taking care to not resuspend beads 6 14 After the second wash dry beads at room temperature for 5 minutes 6 15 Elute cDNA in 24 uL of 10 mM Tris HCl Remove tubes from the magnet and thoroughly resuspend the beads with the 10 mM Tris HCl 6 16 Place cDNA bead solution ba
18. thesis Step 2 First Strand cDNA Synthesis 2 1 Gently open the First Strand cDNA Synthesis SA0002 foil pouch by tearing along the indents located at the top of the silver package 2 2 Remove the purple 8 tube strip Each tube in the strip provides a single reaction 2 3 Centrifuge briefly to ensure lyophilized material is in the bottom of the tube 2 4 Ifyou would like to use fewer than eight reactions detach the appropriate number of tubes carefully using clean scissors or a new razor blade Store the remaining unused tubes in the sealed foil pouch with the desiccant provided at 4 C 2 5 Place the First Strand cDNA Synthesis tubes on ice and transfer 20 uL of the Random Priming mixture Step 1 9 to the lyophilized First Strand cDNA Synthesis pellet and mix well by pipetting up and down Spin briefly to collect contents at the bottom of the tube 2 6 Place the tubes into a thermal cycler with a heated lid set to gt 100 C and incubate as follows Incubation Incubation Step Temperature Time 1 25 C 10 min 2 42 C 30 min 3 80 C 20 min 4 4 C Hold 2 7 Remove the PCR tubes from the thermal cycler and place on ice 3 1 Gently open the Second Strand cDNA Synthesis SA0003 foil pouch by tearing along the indents located at the top of the silver package 6 Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER gt by enzymatics Sii 3 3 3 4 3 5 3 6 St
19. tion a reference sample sheet is available for download at http www enzymatics com archer The reference sample sheet can be modified with the appropriate index sequence tags and reagent tag and uploaded to the MiSeq for the run Level Read Length R1 Read 1 151 R2 Index Read 1 8 R3 Index Read 2 8 R4 Read 2 151 loading conditions below 8 3 1 Starting from a 2 nM pool 8 3 1 1 Mix 10 uL 2 nM library pool 10 uL 0 2 N NaOH in 1 5 mL microcentrifuge tube vortex briefly Archer Universal RNA Fusion Detection v1 for Illumina 9 Platform IFU AK0024 8 Rev A ARCHER s DY er azyma ICS S 8 3 1 2 Incubate for 5 min at room temperature 8 3 1 3 Add 980 uL ice cold Hyb buffer this comes with the MiSeq cartridge 8 3 1 4 This makes a 20 pM stock vortex briefly to mix 8 3 1 5 Mix 500 uL Hyb Buffer 500 uL 20 pM library in new 1 5 mL microcentrifuge tube and vortex briefly 10 pM Library stock 8 3 1 6 Mix 900 uL 10 pM library stock 100 uL 10 pM denatured PhiX and vortex briefly 8 3 1 7 This creates the final loading pool of 10 pM 10 PhiX 8 3 1 8 Add the entire 1000 uL to the MiSeq cartridge and start the run 8 3 2 Starting from a 4 nM pool 8 3 2 1 Mix 10 uL 4 nM library pool 10 uL 0 2 N NaOH in 1 5 mL microcentrifuge tube vortex briefly 8 3 2 2 Incubate for 5 min at room temperature 8 3 2 3 Add 980 uL ice cold Hyb buffer this comes with the MiSeq cartridge 8 3 2 4 This makes a 40 p
Download Pdf Manuals
Related Search