Ultra Low Input mRNA-Seq Sample Preparation - Support
Contents
1. 2 Amplify the PCR tube in the thermal cycler with the lid closed a 98 C for 30 seconds b 15 cycles of 98 C for 10 seconds 60 C for 30 seconds 72 C for 30 seconds c 72 C for 5 minutes d Hold at 4 C 3 Transfer 50 ul of the sample to each well of a new 96 well V bottom plate 4 Vortex the AMPure XP Beads until they are well dispersed then add 80 ul of the mixed AMPure XP Beads to each well of the 96 well V bottom plate containing 50 ul of the PCR amplified library Gently pipette the entire volume up and down 10 times to mix thoroughly 5 Incubate the plate at room temperature for 8 minutes 6 Place the plate on the magnetic stand at room temperature for 5 minutes or until the liquid appears clear 7 Remove and discard all of the supernatant from each well of the plate Take care not to disturb the beads Change the tip after each sample 9 NOTE tt Leave the plate on the magnetic stand while performing the following 80 EtOH wash steps 8 10 8 With the plate remaining on the magnetic stand add 180 ul of freshly prepared 80 EtOH to each well of the plate without disturbing the beads 9 Incubate the plate at room temperature for at least 30 seconds then remove and discard all of the supernatant from each well Take care not to disturb the beads Change the tip after each sample 10 Repeat steps 8 and 9 once for a total of two 80 EtOH washes 11 Remove the plate from the magnetic stand and let the plate stand at2. PCR tube Multiply each volume by the number of samples being prepared Prepare 10 extra reagent mix if you are preparing multiple samples Reagent Volume ul Eluted DNA 32 Klenow Buffer 5 1mM dATP 10 Klenow exo 3 Total Volume per Sample 50 Incubate the sample on a thermal cycler for 30 minutes at 37 C Transfer 50 ul of the sample to each well of a new 96 well V bottom plate Vortex the AMPure XP Beads until they are well dispersed then add 90 ul of the mixed AMPure XP Beads to each well of the 96 well V bottom plate containing the reaction mix Gently pipette the entire volume up and down 10 times to mix thoroughly Incubate the plate at room temperature for 8 minutes Place the plate on the magnetic stand at room temperature for 5 minutes or until the liquid appears clear Remove and discard all of the supernatant from each well of the plate Take care not to disturb the beads Change the tip after each sample Part 11322751 Rev B 10 11 12 13 14 15 16 Page 12 9 NOTE q Leave the plate on the magnetic stand while performing the following 80 EtOH wash steps 9 11 With the plate remaining on the magnetic stand add 180 ul of freshly prepared 80 EtOH to each well of the plate without disturbing the beads Incubate the plate at room temperature for at least 30 seconds then remove and discard all of the supernatant from each well Take care not to disturb the beads Change the tip after
3. Part 11322751 Rev B 10 11 12 13 14 15 16 Page 10 Vortex the AMPure XP Beads until they are well dispersed then add 180 ul of the mixed AMPure XP Beads to each well of the 96 well V bottom plate containing the reaction mix Gently pipette the entire volume up and down 10 times to mix thoroughly Incubate the plate at room temperature for 8 minutes Place the plate on the magnetic stand at room temperature for 5 minutes or until the liquid appears clear Remove and discard all of the supernatant from each well of the plate Take care not to disturb the beads Change the tip after each sample 9 NOTE q Leave the plate on the magnetic stand while performing the following 80 EtOH wash steps 9 11 With the plate remaining on the magnetic stand add 180 ul of freshly prepared 80 EtOH to each well of the plate without disturbing the beads Incubate the plate at room temperature for at least 30 seconds then remove and discard all of the supernatant from each well Take care not to disturb the beads Change the tip after each sample Repeat steps 9 and 10 once for a total of two 80 EtOH washes Remove the plate from the magnetic stand and let the plate stand at room temperature for 15 minutes to dry Resuspend the dried pellet in each well with 32 ul QIAGEN EB Gently pipette the entire volume up and down 10 times to mix thoroughly Change the tip after each sample Incubate the plate at room temperature
4. each sample Repeat steps 9 and 10 once for a total of two 80 EtOH washes Remove the plate from the magnetic stand and let the plate stand at room temperature for 15 minutes to dry Resuspend the dried pellet in each well with 19 ul QIAGEN EB Gently pipette the entire volume up and down 10 times to mix thoroughly Change the tip after each sample Incubate the plate at room temperature for 2 minutes Place the plate on the magnetic stand at room temperature for 2 minutes or until the liquid appears clear Transfer 19 ul of the clear supernatant from each well of the plate to a new 0 2 ml nuclease free thin wall PCR tube Change the tip after each sample SAFE STOPPING POINT If you do not plan to proceed to Ligate Adapters immediately the protocol can be safely stopped here If you are stopping transfer the sample to a 1 5 ml nuclease free non sticky microcentrifuge tube and store it at 15 to 25 C for one day Part 11322751 Rev B Page 13 Ligate Adapters This process ligates adapters to the ends of the DNA fragments The reaction adds distinct sequences to the 5 and 3 ends of each strand in the genomic fragment Later in the workflow additional sequences are added by tailed primers during PCR These additional sequences are necessary for library amplification on the flow cell during cluster formation User Supplied Consumables 0 2 ml Nuclease free thin wall PCR tubes 3 96 well V bottom plate 500 pl A
5. for 2 minutes Place the plate on the magnetic stand at room temperature for 2 minutes or until the liquid appears clear Transfer 32 ul of the clear supernatant from each well of the plate to a new 0 2 ml nuclease free thin wall PCR tube Change the tip after each sample SAFE STOPPING POINT If you do not plan to proceed to Adenylate 3 Ends immediately the protocol can be safely stopped here If you are stopping transfer the sample to a 1 5 ml nuclease free non sticky microcentrifuge tube and store it at 15 to 25 C for one day Part 11322751 Rev B Page 11 Adenylate 3 Ends A single A nucleotide is added to the 3 ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction A corresponding single T nucleotide on the 3 end of the adapter provides a complementary overhang for ligating the adapter to the fragment This strategy ensures a low rate of chimera concatenated template formation User Supplied Consumables 0 2 ml Nuclease free thin wall PCR tubes 2 1 5 ml nuclease free non sticky microcentrifuge tube 96 well V bottom plate 500 pl AMPure XP Beads Freshly Prepared 80 Ethanol EtOH MicroAmp clean adhesive film Paired End Sample Prep Kit content e 1mM dATP e Klenow Buffer e Klenow exo QIAGEN EB Procedure 1 2 Preheat a thermal cycler to 37 C Prepare the following reaction mix in a new 0 2 ml nuclease free thin wall
6. of the plate Take care not to disturb the beads Change the tip after each sample 9 NOTE q Leave the plate on the magnetic stand while performing the following 80 EtOH wash steps 9 11 With the plate remaining on the magnetic stand add 180 ul of freshly prepared 80 EtOH to each well of the plate without disturbing the beads Incubate the plate at room temperature for at least 30 seconds then remove and discard all of the supernatant from each well Take care not to disturb the beads Change the tip after each sample Repeat steps 9 and 10 once for a total of two 80 EtOH washes Remove the plate from the magnetic stand and let the plate stand at room temperature for 15 minutes to dry Resuspend the dried pellet in each well with 23 ul QIAGEN EB Gently pipette the entire volume up and down 10 times to mix thoroughly Change the tip after each sample Incubate the plate at room temperature for 2 minutes Place the plate on the magnetic stand at room temperature for 2 minutes or until the liquid appears clear Transfer 23 ul of the clear supernatant from each well of the plate to a new 0 2 ml nuclease free thin wall PCR tube Change the tip after each sample SAFE STOPPING POINT If you do not plan to proceed to Enrich DNA Fragments immediately the protocol can be safely stopped here If you are stopping transfer the sample to a 1 5 ml nuclease free non sticky microcentrifuge tube and store it at 15 to 25 C for one
7. A amplified material by running 1 ul of the PCR product of 12 ul purified with AMPure XP Beads on a Agilent Technologies 2100 Bioanalyzer using a Agilent High Sensitivity DNA chip Contaminated products should be discarded i NOTE q Reference the Clontech SMARTer Ultra Low RNA Kit User Manual Figure 2 Agilent Profile Clean Sample FU 140 3 100 200 309 400 S700 2000 10380 fp Figure 3 Agilent Profile Contaminated Sample FU sanol 3 3 100 200 0 400 600 1000 3000 10380 Part 11322751 Rev B Page 6 Figure 4 Agilent Profile Negative Control Input DNA Library Quantification Illumina recommends a minimum of 2 ng DNA per library for the Ultra Low Input mRNA Seq Sample Prep protocol The ultimate success of the library preparation strongly depends on using an accurately quantified amount of input DNA Therefore correct quantification of the DNA library is essential Quantify the concentration of the DNA library purified with AMPure XP Beads by manually integrating a peak between 400 9000 bp of the Bioanalyzer trace obtained from Input DNA Library Quality and multiplying the concentration by 12 ul to determine the total amount of library Part 11322751 Rev B Page 7 Consumables and Equipment Check to ensure that you have all of the necessary user supplied consumables and equipment before proceeding to sample preparation Table 1 User Supplied Consumables Consumable 0 2 ml n
8. MPure XP Beads Freshly Prepared 80 Ethanol EtOH MicroAmp clean adhesive film Paired End Sample Prep Kit content e DNA Ligase Buffer 2X e Ultra pure water e PE Adapter Oligo Mix e T4DNA Ligase e QIAGEN EB Procedure 1 Prepare a dilution of the paired end adapter in ultra pure water in a new 0 2 ml nuclease free thin wall PCR tube Covaris Input DNA ng Adapter Water Dilution 8 50 1 9 4 8 1 14 lt 1 4 1 19 2 Prepare the following reaction mix in a new 0 2 ml nuclease free thin wall PCR tube Multiply each volume by the number of samples being prepared Prepare 10 extra reagent mix if you are preparing multiple samples Reagent Volume ul Eluted DNA 19 DNA Ligase Buffer 2X 25 PE Adapter Oligo Mix diluted 1 T4 DNA Ligase 5 Total Volume per Sample 50 3 Incubate the sample at room temperature for 15 minutes 4 Transfer 50 ul of the sample to each well of a new 96 well V bottom plate Part 11322751 Rev B 10 11 12 13 14 15 16 Page 14 Vortex the AMPure XP Beads until they are well dispersed then add 80 ul of the mixed AMPure XP Beads to each well of the 96 well V bottom plate containing the reaction mix Gently pipette the entire volume up and down 10 times to mix thoroughly Incubate the plate at room temperature for 8 minutes Place the plate on the magnetic stand at room temperature for 5 minutes or until the liquid appears clear Remove and discard all of the supernatant from each well
9. Ultra Low Input mRNA Seq Preparing Samples for the Illumina Sequencing Platform FOR RESEARCH USE ONLY Introduction 2 Sample Prep Workflow 3 Best Practices 4 DNA Input Recommendations 5 Consumables and Equipment 7 SMARTer Ultra Low RNA Protocol 8 Perform End Repair 9 Adenylate 3 Ends 11 Ligate Adapters 13 Enrich DNA Fragments 15 Validate Library 18 Part 11322751 Rev B Pagel Page 2 Introduction This protocol explains how to use the Tlumina Paired End Sample Preparation Kit to convert the sheared DNA output from the Clontech SMARTer Ultra Low RNA Kit for Illumina Sequencing into a library of template molecules suitable for subsequent cluster generation and high throughput DNA sequencing Sheared PCR amplified cDNA fragments produced using the SMARTer Ultra Low RNA Kit are processed through end repair procedures the addition of a single A base and the ligation of the adapters The products are then purified and PCR enriched to create the final cDNA library Part 11322751 Rev B Page 3 Sample Prep Workflow The following figure illustrates the Ultra Low Input mRNA Seq Sample Preparation protocol Figure 1 Ultra Low Input mRNA Seq Sample Preparation Workflow Covaris Sheared DNA output from SMARTer Ultra Low RNA Kit Repair Ends Adenylate 3 Ends Ligate Adapters 4 PCR Amplification Part 11322751 Rev B Page 4 Best Practices When preparing libraries for sequenci
10. day Part 11322751 Rev B Page 15 Enrich DNA Fragments This process uses PCR to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library for accurate quantification PCR is performed with two primers that anneal to the ends of the adapters The number of PCR cycles should be minimized to avoid skewing the representation of the library The PCR amplification process performs four key functions Add Sequences Additional sequences are added to the ends of the adapters so that the final amplified templates contain sequences to enable hybridization with primers bound to the flow cell surface for cluster generation Enrich Fragments PCR enriches fragments that have adapters ligated on both ends Fragments with only one or no adapters on their ends are by products of inefficiencies in the ligation reaction Neither species can be used to make clusters as fragments without any adapters cannot hybridize to surface bound primers in the flow cell and fragments with an adapter on only one end can hybridize to surface bound primers but cannot form clusters However the presence of these incomplete ligation products can lead to an overestimation of the final quantity of library DNA if spectrophotometric or fluorometric quantification is performed In contrast these ligation products are not quantified by qPCR Enrich Templates PCR enriches templates that include the
11. ill be of varying lengths and ends may or may not be phosphorylated This step converts the overhangs resulting from fragmentation into blunt ends using the T4 DNA Polymerase and Klenow Enzyme The 3 to 5 exonuclease activity of these enzymes removes 3 overhangs and the polymerase activity fills in the 5 overhangs In addition T4 PNK in this reaction phosphorylates the 5 ends of the DNA fragments User Supplied Consumables 0 2 ml nuclease free thin wall PCR tubes 2 1 5 ml nuclease free non sticky microcentrifuge tube 96 well V bottom plate 500 pl AMPure XP Beads Covaris Sheared DNA output from SMARTer Ultra Low RNA Kit Freshly Prepared 80 Ethanol EtOH MicroAmp clean adhesive film Paired End Sample Prep Kit content e 10 mM dNTP Mix e Klenow Enzyme e T4 DNA Ligase Buffer with 10 mM ATP e T4 DNA Polymerase e T4PNK e QIAGEN EB Procedure 1 Preheat a thermal cycler to 20 C 2 Prepare the following reaction mix in a new 0 2 ml nuclease free thin wall PCR tube Multiply each volume by the number of samples being prepared Prepare 10 extra reagent mix if you are preparing multiple samples Reagent Volume ul Covaris Sheared DNA 2 10 ng 75 T4 DNA Ligase Buffer with 10 mM ATP 10 10 mM dNTP Mix 4 T4 DNA Polymerase 5 Klenow Enzyme 1 T4 PNK 5 Total Volume per Sample 100 3 Incubate the sample on a thermal cycler for 30 minutes at 20 C 4 Transfer 100 ul of the sample to each well of a new 96 well V bottom plate
12. ing a multichannel pipette change the tips after each column Let the mixed samples incubate for 15 minutes at room temperature for maximum recovery When aspirating the cleared solution from the reaction plate and wash step it is important to keep the plate on the magnetic stand and to not disturb the separated magnetic beads Aspirate slowly to prevent the beads from sliding down the sides of the wells and into the pipette tips For the wash steps prepare fresh 80 ethanol Eighty percent ethanol tends to absorb water from the air therefore fresh 80 ethanol should be prepared for optimal results Be sure to remove all of the ethanol from the bottom of the wells as it may contain residual contaminants Remove the reaction plate from the magnetic stand and let it air dry at room temperature Allow for the complete evaporation of residual ethanol as it impacts the performance of the subsequent reactions Illumina recommends at least 15 minutes drying time but a longer drying time may be required Use the QIAGEN EB for DNA elution gently pipette up and down 10 times making sure the liquid comes in contact with the beads and that the beads are resuspended homogeneously Part 11322751 Rev B Page 5 DNA Input Recommendations Input DNA Library Quality High quality SMARTer Ultra Low RNA amplified material is essential for the success of the Ultra Low Input mRNA Seq Sample Prep Verify the size distribution of the SMARTer Ultra Low RN
13. ng you should always adhere to good molecular biology practices Liquid Handling Good liquid handling measures are essential particularly when quantifying libraries or diluting concentrated libraries for making clusters Small differences in volumes 0 5 ul can sometimes give rise to very large differences in cluster numbers 100 000 Small volume pipetting can be a source of potential error in protocols that require generation of standard curves such as PicoGreen assays or qPCR or those that require small but precise volumes such as the Agilent BioAnalyzer If small volumes are unavoidable then due diligence should be taken to ensure that pipettes are correctly calibrated Ensure that pipettes are not used at the volume extremes of their performance specifications Care should be taken because solutions of high molecular weight dsDNA can be viscous and not evenly dispersed resulting in aliquot measurements that are not representative of the true concentration of the solution AMPure Bead Handling The following indicates the appropriate handling methods when working with Agencourt AMPure XP Beads Prior to use allow the beads to come to room temperature Immediately prior to use vortex the beads until they are well dispersed The color of the liquid should appear homogeneous After adding the beads to the reaction mix the solution thoroughly by pipetting up and down 10 times Change the tips for each sample or when us
14. non template A nucleotide added during the A tailing process and therefore eliminates adapter dimers This is accomplished by using proprietary modified primers that are completely resistant to the 3 5 exonuclease activity of the Phusion Finnzymes Oy polymerase used for PCR These primers reach all the way to the non templated A before the start of the genomic sequence If the A is not present as in the case for an adapter dimer then the terminal T on the primer will mismatch and not extend Provides Material PCR provides enough material to enable reliable quantification of the final library if spectrophotometric or fluorometric methods are used User Supplied Consumables e 0 2 ml nuclease free thin wall PCR tube e 1 5 ml nuclease free non sticky microcentrifuge tube e 96 well V bottom plate 500 ul e AMPure XP Beads e Freshly Prepared 80 Ethanol EtOH e MicroAmp clean adhesive film e Paired End Sample Prep Kit content e Phusion DNA Polymerase Finnzymes Oy e PCR Primer PE 1 0 e PCR Primer PE 2 0 Part 11322751 Rev B Page 16 Procedure 1 Prepare the following reaction mix in a new 0 2 ml nuclease free thin wall PCR tube on ice Multiply each volume by the number of samples being prepared Prepare 10 extra reagent mix if you are preparing multiple samples Reagent Volume ul DNA 23 Phusion DNA Polymerase 25 PCR Primer PE 1 0 1 PCR Primer PE 2 0 1 Total Volume per Sample 50
15. room temperature for 15 minutes to dry Part 11322751 Rev B 12 13 14 15 Page 17 Resuspend the dried pellet in each well with 15 ul QIAGEN EB Gently pipette the entire volume up and down 10 times to mix thoroughly Change the tip after each sample Incubate the plate at room temperature for 2 minutes Place the plate on the magnetic stand at room temperature for 2 minutes or until the liquid appears clear Transfer 15 ul of the clear supernatant from each well of the plate to a new 1 5 ml nuclease free non sticky microcentrifuge tube and store it at 20 C Part 11322751 Rev B Page 18 Validate Library Illumina recommends performing the following quality control analysis on your sample library to quantify the DNA concentration 1 Load 1 ul of the resuspended construct on an Agilent Technologies 2100 Bioanalyzer using a DNA specific chip such as the Agilent DNA 1000 Figure 5 Final Ultra Low Input mRNA Seq Sample Prep Library Bioanalyzer Trace 2 Check the size purity and concentration of the sample The final product should be a distinct band at approximately 250 bp Part 11322751 Rev B
16. uclease free thin wall PCR tubes 1 5 ml nuclease free non sticky microcentrifuge tubes 96 well V bottom plates 500 ul Agencourt AMPure XP PCR Purification Kit 60 ml Covaris sheared DNA 75 ul Freshly Prepared 80 Ethanol MicroAmp Clean Adhesive Film Nuclease free water Paired End Sample Prep Kit QIAGEN EB Buffer Table 2 User Supplied Equipment Equipment Magnetic stand 96 Thermal cycler Part 11322751 Rev B Supplier USA Scientific part 1402 4700 USA Scientific part 1415 2600 VWER catalog 47743 996 Beckman Coulter Genomics part 63881 User supplied output from SMARTer Ultra Low RNA Kit Clontech part 634935 General lab supplier Applied Biosystems part 4306311 General lab supplier Illumina catalog PE 102 1001 10 samples or PE 102 1002 40 samples QIAGEN part 19086 Supplier Ambion part AM10027 General lab supplier Page 8 SMARTer Ultra Low RNA Protocol Prior to Ultra Low Input mRNA Seqg Sample Preparation you must first PCR amplify and fragment the cDNA library using the SMARTer Ultra Low RNA Kit Reference the Clontech SMAR Ter Ultra Low RNA Kit User Manual To ensure high quality DNA input for this protocol see DNA Input Recommendations on page 5 Part 11322751 Rev B Page 9 Perform End Repair DNA fragmentation by physical methods produces heterogeneous ends comprising a mixture of 3 overhangs 5 overhangs and blunt ends The overhangs w
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