illumina TruSeq DNA PCR-Free Sample Prep. (LS) Protocol

1

Part# 15036187 Revision A January 2013 Performed using the TruSeq DNA PCR-Free Sample Preparation Kit (A cat#FC-121-3001, B cat#FC-121-3002)

Fragment DNA Fragmentation will be conducted using the Covaris S2 Ultrasonicator. NOTE: Thaw frozen Resuspension Buffer (10mM Tris pH 8.5) to RT˚ for use during protocol. After use place Resuspension Buffer at 4˚C for long term storage. NOTE: Turn on the Covaris system >30 minutes before starting protocol. De-gas and pre-chill the deionized H2O to a temperature of 3˚C to 6˚C. The fragmentation procedure may be started when 6˚C is reached.

Library Insert Size Options Insert Size 350bp 550bp Input DNA per sample 1ug 2ug Recommended Read Length ≤ 2x101bp ≤ 2x151bp* * Read lengths greater than 2x151bp will produce a significantly higher percentage of overlapping reads.

gDNA Fragmentation 1. Dilute 1ug or 2ug of gDNA to 55ul total volume in Resuspension Buffer for a final concentration of 20ng/ul or 40ng/ul. 2. Transfer 52.5ul of diluted DNA from the DNA labeled plate to a Covaris tube. ADD VOLUME SLOWLY AVOIDING BUBBLES 3. Fragment the DNA using the following settings: NOTE: For the DNA PCRfree create the following programs for Covaris S2 - 350bp DNA PCRfree micro_070113 - 550bp DNA PCRfree micro_070113 Covaris S2 Duty Cycle Intensity Bursts per second Duration Mode Power Temperature

350bp Insert 10% 5.0 200 45 seconds Frequency Sweeping 23W 5.5˚C – 6.0˚C

550bp Insert 10% 2.0 200 45 seconds Frequency Sweeping 9W 5.5˚C – 6.0˚C

NOTE: Make sure Covaris tube is properly positioned with the correct water level before initiating sonication. 4. Seal the Covaris tube and centrifuge at 600xg for 1 minute using an appropriate adaptor for the microcentrifuge rotor. 5. Transfer 50ul of the fragmented DNA from each Covaris tube to the corresponding well of a new 0.3ml PCR plate labeled with the CSP barcode.

Clean Up of Fragmented DNA NOTE: Remove Sample Purifcation Beads and Resuspension Buffer from 4o and -20o. Warm beads to RT o. Place thawed resuspension buffer on ice. NOTE: Apply IMP barcode to new 0.3ml PCR plate.

1. Vortex the Sample Purification Beads for at least 1 minute or until beads are well dispersed.

2 2. Add 80 ul of Sample Purification Beads to each well of the CSP plate containing 50 ul of fragmented gDNA. Gently pipette 10x to mix thoroughly. NOTE: Keep Sample Purification Beads at RT o for later use in the protocol. 3. Incubate CSP plate at RTo for 5 minutes. 4. Place CSP plate on magnetic stand for at least 8 minutes at RTo or until liquid clears. 5. Remove and discard 125 ul of supernatant. NOTE: Leave IMP plate on the magnetic stand while performing the following 80% ETOH washes. NOTE: Prepare fresh 80% ETOH for all subsequent washing procedures. 6. Add 200ul 80% ETOH to each well of the CSP plate without disturbing the bead pellet. 7. Incubate the CSP plate at RT˚ for 30 seconds then remove and discard all supernatant from each well. DO NOT DISTURB PELLET. 8. Repeat ETOH wash for a total of two 80% ETOH washes. 9. Let CSP plate stand at RT˚ for 10 min to dry. Remove and discard any remaining EtOH with a 10ul pipette. 10. While keeping the CSP plate on the magnetic stand, add 52.5 ul of Resuspension Buffer to each well, and then remove plate from magnetic stand. 11. Resuspend the dried pellet in each well by gently pipetting the entire volume up and down 10x to mix thoroughly. 12. Incubate the CSP plate at RT˚ for 2 minutes. 13. Place the CSP plate on the magnetic stand at RT˚ for 5 minutes or until liquid appears clear. 14. Transfer 50 ul of the clear supernatant from each well of the CSP plate to the corresponding well of a new 0.3ml PCR plate with an IMP barcode. DO NOT DISTURB PELLET. 15. Proceed immediately to Perform End Repair and Size Selection.

End Repair NOTE: Thaw the following: End Repair Control, End Repair Mix 2. Mix well and centrifuge briefly. Place on ice. NOTE: Make sure Sample Purification Beads and Resuspension Buffer are at room temperature. NOTE: Pre-heat thermal cycler to 30˚C. NOTE: Apply an ALP barcode to a new 0.3ml PCR plate. NOTE: Apply a CEP barcode to new 0.3ml PCR plate. 1. Add 10ul of the End Repair Control to each sample in the IMP plate containing 50ul of fragmented DNA. Substitute 10ul Resuspension Buffer if End Repair Control is not used. 2. Add 40ul of End Repair Mix 2 to each well of the IMP plate. Adjust volume of pipette to 100ul and pipette up and down 10x to mix. 3. Seal the IMP plate with a Microseal ‘B’ adhesive seal. 4. Incubate the IMP plate in a pre-heated thermal cycler using the following profile: ERP

30˚C 4oC

30 min hold

NOTE: Use heated lid at 100oC

5. Remove IMP plate from thermal cycler when program reaches 4oC. 6. Remove adhesive seal from the IMP plate.

3

IMP Clean Up and Size Selection Remove Large DNA Fragments

1. Vortex Sample Purification Beads for at least 1 minute or until they are well dispersed. 2. Add the Sample Purification Beads and PCR grade water to a new 15 ml conical tube to create a diluted bead mixture of 160 ul per 100 ul of end-repaired sample using the following formula, which includes a 15% excess for multiple samples: NOTE: For less than 6 samples use a 1.7 ml microcentrifuge tube to prepare diluted bead mixture.

Diluted Bead Mixture for 350 bp Insert Size Formula Sample Purification Beads PCR grade water

# of samples X 109.25 ul # of samples X 74.75 ul

Example Amount per 12 samples 1311 ul 897 ul

Diluted Bead Mixture for 550 bp Insert Size Formula Sample Purification Beads PCR grade water

# of samples X 92 ul # of samples X 92 ul

Example Amount per 12 samples 1104 ul 1104 ul

3. Vortex diluted bead mixture for 5 seconds to make sure the beads are evenly dispersed. NOTE: Vortex the diluted bead mixture frequently by vortexing the mixture after processing four samples (for single channel pipette) or four columns (for multichannel). 4. Add 160 ul of the diluted bead mixture to each well of the IMP plate containing 100 ul of end repaired sample. Gently pipette the entire volume up and down 10x to mix thoroughly. NOTE: Aspirate the diluted bead mixture very slowly and dispense it very slowly due to the viscosity of the solution. Changes in the volume of the diluted bead mixture affect the insert size of your library. 5. Incubate IMP plate at RT˚ for 5 min. 6. Place IMP plate on the magnetic stand at RT˚ for 5 min or until liquid appears clear. NOTE: TRANSFER, DO NOT DISCARD, THE SUPERNATANT!!! It contains the DNA of interest. 7. Transfer 125 ul of the supernatant, containing the DNA of interest, two times from each well of the IMP plate to the corresponding labeled CEP plate. Each CEP plate well will contain a total of 250 ul of DNA of interest. Take care not to disturb beads. NOTE: TRANSFER, DO NOT DISCARD, THE SUPERNATANT!!! It contains the DNA of interest. 8. Discard the IMP plate containing the beads. 9. Discard any remaining diluted bead mixture.

Remove Small DNA Fragments 1. Vortex Sample Purification Beads for at least 1 minute or until they are well dispersed. NOTE: Vortex the Sample Purification Beads frequently by vortexing the mixture after processing four samples (for single channel pipette) or four columns (for multichannel).

4 2. Add 30 ul of undiluted Sample Purification Beads to each well of the CEP plate containing 250 ul of supernatant with DNA of interest. Gently pipette up and down 10x to mix thoroughly. NOTE: Aspirate the diluted bead mixture very slowly and dispense it very slowly due to the viscosity of the solution. Changes in the volume of the diluted bead mixture affect the insert size of your library. 3. Incubate CEP plate at RTo for 5 minutes. 4. Place CEP plate on the magnetic stand at RT˚ for 5 min or until liquid appears clear. 5. Remove and discard 138 ul of supernatant from each well of the CEP plate. Take care not to disturb beads. 6. Repeat step 5 once ( 276ul discarded total) . NOTE: Leave IMP plate on the magnetic stand while performing the following 80% ETOH washes. 7. Add 200ul 80% ETOH to each well of the CEP plate without disturbing the bead pellet. 8. Incubate the CEP plate at RT˚ for 30 seconds then remove and discard all supernatant from each well. DO NOT DISTURB PELLET. 9. Repeat ETOH wash for a total of two 80% ETOH washes. 10. Let CEP plate stand at RT˚ for 10 min to dry. Remove and discard any remaining EtOH with a 10 ul pipette. 11. While keeping the CEP plate on the magnetic stand, add 17.5 ul of Resuspension Buffer to each well, and then remove plate from magnetic stand. 12. Resuspend the dried pellet in each well by gently pipetting the entire volume up and down 10x to mix thoroughly. 13. Incubate the CEP plate at RT˚ for 2 minutes. 14. Place the CEP plate on the magnetic stand at RT˚ for 5 minutes or until liquid appears clear. 15. Transfer 15 ul of the clear supernatant from each well of the CEP plate to the corresponding well of a new 0.3ml PCR plate with an ALP barcode. NOTE: SAFE STOPPING POINT – seal the ALP plate with a Microseal ‘B’ adhesive seal and store at -25˚C for 7 days.

Adenylate 3’ Ends NOTE: Thaw the following at RT˚: A-Tailing Control, A-Tailing Mix. Centrifuge briefly when thawed. NOTE: Warm Resuspension Buffer to RT˚ before use NOTE: Thaw ALP plate at RT˚ if necessary and centrifuge briefly at 280xg for 1 minute. Remove adhesive seal. NOTE: Pre-heat thermal cycler to 37˚C. 1. Add 2.5ul of the A-Tailing Control to each well of the ALP plate. Substitute 2.5ul Resuspension Buffer if A-Tailing Control is not used. 2. Add 12.5ul of A-Tailing Mix to each well of the ALP plate. 3. Adjust the pipette to 30ul and gently pipette the entire volume up and down 10x to mix. Change tip after each sample. 4. Seal the ALP plate with a Microseal ‘B’ adhesive seal. 5. Incubate the ALP plate in a pre-heated thermal cycler using the following profile: ATAIL70

37˚C 70oC 4oC 4oC

30 min 5 min 5min HOLD

NOTE: Use heated lid at 100oC

6. Remove the ALP from the thermal cycler after it has been at 4oC for 5 minutes then proceed immediately to Ligate Adapters.

5 Ligate Adapters NOTE: Thaw the following at RT˚: Appropriate DNA Adapter Index tubes (AD001-AD012), Stop Ligation Buffer, Ligation Control. NOTE: Remove the Sample Purification Beads and Resuspension Buffer from 4˚C and let warm to RT˚ for 30 minutes. NOTE: Pre-heat thermal cycler to 30˚C. NOTE: Apply a CAP barcode label to a new 0.3ml PCR plate. NOTE: Apply a TSP1 barcode label to a new 0.ml PCR plate. Apply a DAP barcode label to a new 0.3ml PCR plate if using the HT kit. NOTE: See Illumina TruSeq DNA PCR-Free Sample Preparation Guide for the use of a DAP plate in the HT kit. 1. Mix well and briefly centrifuge the thawed DNA Adapter Index tubes, Ligase Control and Stop Ligation Buffer tubes at 600xg. 2. Briefly centrifuge ALP plate at 280xg. Remove the adhesive seal from the ALP plate. 3. Add 2.5ul of the Ligase Control to each well of the ALP plate. Substitute 2.5ul Resuspension Buffer if A-Tailing Control is not used. 4. Leave the Ligation Mix 2 in the Stratacooler during use. 5. Add 2.5ul of Ligation Mix 2 to each well of the ALP plate. 6. Immediately return the cooler with Ligation Mix 2 back to -25˚C storage after use. 7. Add 2.5ul of the appropriate DNA Adapter Index to each well of the ALP plate. 8. Adjust the pipette to 37.5ul and pipette the entire volume up and down 10x to mix. 9. Seal the ALP plate with a Microseal ‘B’ adhesive seal. 10. Incubate the ALP plate in a pre-heated thermal cycler at 30˚C for 10 minutes using the following profile: LIG30

30˚C

10 min

4oC

HOLD

NOTE: Use heated lid at 100oC

11. Remove the ALP plate from the thermal cycler and remove seal. 12 Add 5ul of Stop Ligation Buffer to each well of the ALP plate to inactivate the ligation. 13. Adjust the pipette to 42.5ul and gently pipette up and down 10x to mix thoroughly.

ALP Clean Up 1. Vortex the Samples Purification Beads until they are well dispersed. NOTE: Vortex the beads frequently. 2. Add 42.5ul of beads to each well of the ALP plate. NOTE: Keep beads at RT˚ for use later in this clean-up procedure. 3. Adjust the pipette to 85ul and pipette the entire volume up and down 10x to mix thoroughly. 4. Incubate the ALP plate at RT˚ for 5 minutes. 5. Transfer the ALP plate to the magnetic stand at RT˚ for 5 minutes or until liquid appears clear. 6. Remove and discard 80ul of the supernatant from each well. NOTE: Leave ALP plate on the magnetic stand while performing the following 80% ETOH washes. 7. With the ALP remaining on the magnetic stand, add 200ul of freshly prepared 80% ETOH to each well. DO NOT DISTURB PELLET. 8. Incubate the ALP plate at RT˚ for 30 seconds. Remove and discard the supernatant from each well avoiding pellet. 9. Repeat wash steps for a total of two 80% ETOH washes.

6 10. Keep the ALP plate on the magnetic stand and allow to air dry at RT˚ for 10 minutes. Remove and discard any remaining EtOH with a 10 ul pipette. 11. While keeping the ALP plate on the magnetic stand, add 52.5 ul of RT˚ Resuspension Buffer to each well, and then remove plate from magnetic stand. 12. Resuspend the dried pellet in each well by gently pipetting the entire volume up and down 10x to mix thoroughly. 13. Incubate the ALP plate at RT˚ for 2 minutes. 14. Place the ALP plate on the magnetic stand at RT˚ for 5 minutes or until liquid appears clear. 15. Transfer 50 ul of the clear supernatant from each well of the ALP plate to the corresponding well of a new 0.3ml PCR plate with a CAP barcode. DO NOT DISTURB PELLET. 16. Vortex the Samples Purification Beads until they are well dispersed NOTE: Vortex the beads frequently. 17. Add 50 ul of beads to each well of the CAP plate for second bead clean up. 18. Adjust the pipette to 85ul and pipette the entire volume up and down 10x to mix thoroughly. 19. Incubate the CAP plate at RT˚ for 5 minutes. 20. Transfer the CAP plate to the magnetic stand at RT˚ for 5 minutes or until liquid appears clear. 21. Remove and discard 95 ul of the supernatant from each well. NOTE: Leave CAP plate on the magnetic stand while performing the following 80% ETOH washes. 22. With the CAP remaining on the magnetic stand, add 200ul of freshly prepared 80% ETOH to each well. DO NOT DISTURB PELLET. 23. Incubate the CAP plate at RT˚ for 30 seconds. Remove and discard the supernatant from each well avoiding pellet. 24. Repeat wash steps for a total of two 80% ETOH washes. 25. Keep the CAP plate on the magnetic stand and allow to air dry at RT˚ for 10 minutes. Remove and discard any remaining EtOH with a 10 ul pipette. 26. While keeping the CAP plate on the magnetic stand, add 22.5 ul of RT˚ Resuspension Buffer to each well, and then remove plate from magnetic stand. 27. Resuspend the dried pellet in each well by gently pipetting the entire volume up and down 10x to mix thoroughly. 28. Incubate the CAP plate at RT˚ for 2 minutes. 29. Place the CAP plate on the magnetic stand at RT˚ for 5 minutes or until liquid appears clear. 30. Transfer 20 ul of the clear supernatant from each well of the CAP plate to an appropriately labeled 1.5ml microfuge tube. DO NOT DISTURB PELLET.

Validate Library NOTE: qPCR must be used to quantify TruSeq DNA PCR-Free Sample Prep libraries. Methods other than qPCR will quantify molecules that do not have adaptors on both ends and will not form clusters. More of these non-clusterable molecules may be present due to the absence of PCR enrichment and quantification by methods other than qPCR may be inaccurate. 1. Perform QC of the amplified library by loading 1ul of library on a DNA1000 or 1 ul of a 1/5 diluted library on the Agilent High Sensitivity DNA chip NOTE: Agilent run is for qualitative purposes only. NOTE: PCR-Free library fragment sizes measured on the Bioanalyzer are substantially larger than would be predicted or derived from sequencing data. This is due to anomalous migration of fragments on the chip due to the presence of certain structural features which would normally be removed if a subsequent PCR-enrichment were performed. 2. Calculate the nM concentration of each library. 3. Transfer 5 ul of each library from the TSP1 plate to a new Midi plate labeled with a DCT barcode and dilute to 2 nM concentration using EB buffer (10mM Tris HCL pH 8.5) supplemented with 0.1% Tween20.

7 4. Pipet up and down with multichannel pipette 10x to mix 5. For non-multiplexed samples that do not require pooling proceed to cluster generation NOTE: Store DCT barcoded MIDI plate with diluted libraries at -20˚C until ready to generate clusters. NOTE: Libraries diluted to 2 nM in EB buffer supplemented 0.1% Tween20 are safe for long term storage at -20˚C

Pooling Libraries (Optional) 1. Apply a PDP barcode label to a new 0.3ml plate (for multiplexing only). 2. Determine the samples to be combined together for each pool. NOTE: Verify that libraries to be pooled have unique indexes!! 3. Transfer 5 ul of each normalized library to be pooled from the DCT plate to one well of the new PDP labeled plate. The total volume of the combines sample libraries will 5x the number of combined samples (6 libraries = 30ul). 4. Pipette the entire volume up and down 10x to thoroughly mix the pooled libraries. 5. Proceed to cluster generation.