TITLE: Gene Expression Dual Index Library Construction
AUTHORS: Heidi Monroe, Nayra Cardenes, Melanie Königshoff, koenigshoffm, Robert Lafyatis

[DESCRIPTION]
The Chromium Single Cell Gene Expression Solution upgrades short read sequencers to deliver a scalable microfluidic platform for 3’ digital gene expression by profiling 500-10,000 individual cells per sample. 

Once cDNA is amplified, enzymatic fragmentation and size selection are used to optimize the cDNA amplicon size. P5, P7, i7 and i5 sample indexes, and TruSeq Read 2 (read 2 primer sequence) are added via End Repair, A-tailing, Adaptor Ligation, and PCR. The final libraries contain the P5 and P7 primers used in Illumina amplification.

This protocol details the gene expression dual index library construction and sequencing.

[BEFORE_START]
Equilibrate to Room temperature (RT) – Fragmentation Buffer (2000091), Adaptor Oligos (2000094), Ligation Buffer (2000092) and Dual Index Plate TT Set A (3000431)
Place on ice– Fragmentation Enzyme (2000090/2000104), DNA Ligase (220110/220131) and Amp Mix (2000047/2000103)
Thaw at 65°C- Cleanup Buffer (2000088)


[STEPS]
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1. Fragmentation, End Repair & A-tailing

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1.1. Prepare a thermal cycler with the following incubation protocol:
 
 

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1.2. Vortex fragmentation buffer. Verify there is no precipitate.

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1.3. Prepare fragmentation mix on ice. Pipette mix and centrifuge briefly.

 Fragmentation Mix
 
 
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1.4. Transfer ONLY 10 µLpurified cDNA sample from pellet cleanup to a tube strip. The remaining 30 µL(75%) cDNA sample can be stored at 4 °C for up to 4320 min or at -20 °C for up to 4 weeks for generating additional 3ʹ Gene Expression libraries.

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1.5. Add 25 µL buffer EB to each sample.

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1.6. Add 15 µL fragmentation mix to each sample.

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1.7. Pipette mix 15× (pipette set to 35 µl)on ice. Centrifuge briefly.

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1.8. Transfer into the pre-cooled thermal cycler (4 °C) and press “SKIP” to initiate the protocol.

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2. Post Fragmentation,End Repair & A-tailing Double Sided Size Selection – SPRIselect:
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2.1. Vortex to resuspend SPRIselect reagent. Add 30 µL SPRIselect (0.6X) reagent to each sample. Pipette mix 15× (pipette set to 75 µl). 
SECTION: Gene Expression Dual Index Library Construction
2.2. Incubate 5 min at Room temperature.

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2.3. Place on the magnet. High until the solution clears. DO NOT discard supernatant.

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2.4. Transfer75 µLsupernatant to a new tube strip.

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2.5. Vortex to resuspend SPRIselect reagent. Add 10 µLSPRIselect reagent (0.8X) to each transferred supernatant. Pipette mix 15× (pipette set to 80 µl).

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2.6. Incubate 5 min at Room temperature.

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2.7. Place on the magnet. High until the solution clears.

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2.8. Remove 80 µL supernatant. DO NOT discard any beads.

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2.9. Add 125 µL 80% ethanol to the pellet. Wait 30 s.

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2.10. Remove the ethanol.

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2.11. Repeat sub-steps 2.9 and 2.10 for a total of 2 washes.

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2.12. Centrifuge briefly. Place on the magnet. Low until the solution clears. Remove remaining ethanol.

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2.13. Remove from the magnet. Add 50.5 µL buffer EB to each sample. Pipette mix 15× (pipette set to 45 µl).

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2.14. Incubate 2 min at Room temperature.

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2.15. Place on the magnet. High until the solution clears.

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2.16. Transfer50 µL sample to a new tube strip.

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3. Adaptor Ligation

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3.1. Prepare adaptor ligation Mix. Pipette mix and centrifuge briefly.
 
Adaptor Ligation Mix:
 
 
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3.2. Add 50 µL adaptor ligation mix to 50 µL sample. Pipette mix 15× (pipette set to 90 µl). Centrifuge briefly.

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3.3. Incubate in a thermal cycler with the following protocol:
 
 

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4. Post Ligation Cleanup – SPRIselect:
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4.1. Vortex to resuspend SPRIselect reagent. Add 80 µLSPRIselect (0.8X) reagent to each sample. Pipette mix 15× (pipette set to 75 µl).

SECTION: Gene Expression Dual Index Library Construction
4.2. Incubate 5 min at Room temperature.

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4.3. Place on the magnet. High until the solution clears. 

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4.4. Remove the supernatant.

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4.5. Add 200 µL 80% ethanol to the pellet. Wait 30 s.

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4.6. Remove the ethanol.

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4.7. Repeat sub-steps 4.5 and 4.6 for a total of 2 washes.

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4.8. Centrifuge briefly. Place on the magnet. Low until the solution clears. 

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4.9. Remove remaining ethanol. Air dry for 2 min.

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4.10. Remove from the magnet. Add 30.5 µL buffer EB to each sample. Pipette mix 15x.

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4.11. Incubate 2 minatRoom temperature.

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4.12. Place on the magnet. Low until the solution clears.

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4.13. Transfer 30 µL sample to a new tube strip.

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5. Sample Index PCR
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5.1. Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10× Sample Index name (PN-3000431 Dual Index Plate TT Set A well ID) used.

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5.2. Add 50 µL Amp mix to 30 µL sample.

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5.3. Add 20 µL of an individual Dual Index TT Set A to each sample and record the well ID used. Pipette mix 5× (pipette set to 90 µl). Centrifuge briefly.

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5.4. Incubate in a thermal cycler with the following protocol:
 
 

 

 
 
 
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5.5. Store at 4 °C for up to 4320 min or proceed to the next step.

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6. Post Sample Index PCR Double Sided Size Selection – SPRIselect
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6.1. Vortex to resuspend SPRIselect reagent. Add 60 µL SPRIselect (0.6X) reagent to each sample. Pipette mix 15× (pipette set to 150 µl).

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6.2. Incubate 5 min at Room temperature.

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6.3. Place on the magnet. High until the solution clears. DO NOT discard supernatant.

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6.4. Transfer 150 µL supernatant to a new tube strip.

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6.5. Vortex to resuspend SPRIselect reagent. Add 20 µL SPRIselect reagent (0.8X)to each transferred supernatant. Pipette mix 15× (pipette set to 150 µl).

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6.6. Incubate 5 min at Room temperature.

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6.7. Place on the magnet. High until the solution clears.

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6.8. Remove 165 µL supernatant. DO NOT discard any beads.

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6.9. Add 200 µL 80% ethanol to the pellet. Wait 30 s.

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6.10. Remove the ethanol.

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6.11. Repeat sub-steps 6.9 and 6.10 for a total of 2 washes.

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6.12. Centrifuge briefly. Place on the magnet. Low until the solution clears. Remove remaining ethanol.

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6.13. Remove from the magnet. Add 35.5 µL buffer EB to each sample. Pipette mix 15× (pipette set to 35 µl).

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6.14. Incubate 2 min at Room temperature.

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6.15. Place on the magnet. Low until the solution clears.

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6.16. Transfer 35 µL sample to a new tube strip.

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6.17. Store at 4 °C for up to 4320 min or at -20 °C for long-term storage.

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7. Post Library Construction QC

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7.1. Run 1 µL sample at 1:10 dilution on an Agilent Bioanalyzer High Sensitivity chip.

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7.2. Determine the average fragment size from the Bioanalyzer trace. This will be used as the insert size for library quantification.

SECTION: Sequencing
8. 3’ Gene Expression Library Sequencing Depth & Run Parameters:
 
 
 
SECTION: Sequencing
9. Once quantified and normalized, the 3’ Gene Expression libraries should be denatured and diluted as recommended for Illumina sequencing platforms.