TITLE: Post GEM–RT Cleanup and cDNA Amplification
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. 

A pool of ~3,500,000 10x Barcodes are sampled separately to index each cell’s transcriptome. It is done by partitioning thousands of cells (or nuclei) into nanoliter-scale Gel Beads-in-emulsion (GEMs), where all generated cDNA share a common 10x Barcode. Dual Indexed libraries are generated and sequenced from the cDNA and 10x Barcodes are used to associate individual reads back to the individual partitions.

After the GEMs Generation and Barcoding, GEMs are broken and pooled fractions are recovered. Silane magnetic beads are used to purify the first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. Barcoded, full-length cDNA is amplified via PCR to generate sufficient mass for library construction.

This protocol details the post GEM-RT cleanup and cDNA amplification, cleanup - SPRIselect and quantification.

[BEFORE_START]
Equilibrate to Room temperature (RT) – Reducing Agent B (2000087), cDNA Primers (2000089) and Dynabeads MyOne SILANE (2000048)
Place on ice – Amp Mix (2000047/2000103)
Thaw at 65°C- Cleanup Buffer (2000088)

[STEPS]
SECTION: Dynabeads
1. Add 125 µL Recovery Agent to each sample at Room temperature. 

 

The resulting biphasic mixture contains Recovery Agent/Partitioning Oil (pink) and aqueous phase (clear), with no persisting emulsion (opaque).

 

SECTION: Dynabeads
2. Slowly remove and discard 125 µLRecovery Agent/Partitioning Oil (pink) from the bottom of the tube. 

 

SECTION: Dynabeads
3. Prepare Dynabeads Cleanup Mix:

 
 
SECTION: Dynabeads
4. Vortex and add 200 µL to each sample. Pipette mix 10x (pipette set to 200 µl).

SECTION: Dynabeads
5. Incubate 10 min at Room temperature .
 

Pipette mix again at ~5 min after start of incubation to resuspend settled beads.

SECTION: Dynabeads
6. Prepare Elution Solution I. Vortex and centrifuge briefly. 

Elution Solution I
 
 
SECTION: Dynabeads
7. At the end of 10 min incubation, place on a 10x Magnetic Separator. High position (magnet.High) until the solution clears.

SECTION: Dynabeads
8. Remove the supernatant (aqueous phase and Recovery Agent).

SECTION: Dynabeads
9. Add 300 µL 80% ethanol to the pellet while on the magnet. Wait 30 s.

SECTION: Dynabeads
10. Remove the ethanol.

SECTION: Dynabeads
11. Add 200 µL 80% ethanol to pellet. Wait 30 s.

SECTION: Dynabeads
12. Remove the ethanol.

SECTION: Dynabeads
13. Centrifuge briefly. Place on the magnet. Low.

SECTION: Dynabeads
14. Remove remaining ethanol. Air dry for 1 min.

SECTION: Dynabeads
15. Remove from the magnet. Immediately add 35.5 µL Elution Solution I.

SECTION: Dynabeads
16. Pipette mix (pipette set to 30 µl) without introducing bubbles.

SECTION: Dynabeads
17. Incubate 2 min at Room temperature.
SECTION: Dynabeads
18. Place on the magnet. Low until the solution clears.

SECTION: Dynabeads
19. Transfer 35 µL sample to a new tube strip.

SECTION: cDNA amplification
20. Prepare cDNA Amplification Mix on ice. Add reagents in the order listed. Vortex and centrifuge briefly. 

cDNA Amplification Reaction Mix
 
 
SECTION: cDNA amplification
21. Add 65 µL cDNA Amplification Reaction Mix to 35 µL sample.

SECTION: cDNA amplification
22. Pipette mix 15x (pipette set to 90 µl). Centrifuge briefly.

SECTION: cDNA amplification
23. Incubate in a thermal cycler with the following protocol:
 
 

SECTION: cDNA amplification
24. Store at 4 °C for up to 4320 min or -20 °C for ≤1 week or proceed to the next step.

SECTION: cDNA Cleanup – SPRIselect:
25. Vortex to resuspend the SPRIselect reagent. Add 60 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 150 µl).

SECTION: cDNA Cleanup – SPRIselect:
26. Incubate 5 min at Room temperature.

SECTION: cDNA Cleanup – SPRIselect:
27. Place on the magnet. High until the solution clears.

SECTION: cDNA Cleanup – SPRIselect:
28. Remove the supernatant.

SECTION: cDNA Cleanup – SPRIselect:
29. Add 200 µL 80% ethanol to the pellet. Wait 30 s.

SECTION: cDNA Cleanup – SPRIselect:
30. Remove the ethanol.

SECTION: cDNA Cleanup – SPRIselect:
31. Repeat steps 29 and 30 for a total of 2 washes.

SECTION: cDNA Cleanup – SPRIselect:
32. Centrifuge briefly and place on the magnet. Low.

SECTION: cDNA Cleanup – SPRIselect:
33. Remove any remaining ethanol. Air dry for 2 min. 

 

SECTION: cDNA Cleanup – SPRIselect:
34. Remove from magnet. Add 40.5 µL Buffer EB. Pipette mix 15x.

SECTION: cDNA Cleanup – SPRIselect:
35. Incubate for 2 min at Room temperature.

SECTION: cDNA Cleanup – SPRIselect:
36. Place the tube strip on the magnet. High until the solution clears.

SECTION: cDNA Cleanup – SPRIselect:
37. Transfer 40 µL sample to a new tube strip.

SECTION: cDNA Cleanup – SPRIselect:
38. Store at 4 °C for up to 4320 min or at -20 °C for up to 4 weeks, or proceed to the next step.

SECTION: cDNA Quantification
39. Run 1 µL sample (Dilution Factor 1:10) on an Agilent Bioanalyzer High Sensitivity chip.