Post GEM–RT Cleanup and cDNA Amplification
Melanie Königshoff, Melanie Königshoff, Nayra Cardenes, Robert Lafyatis, Heidi Monroe
Post GEM-RT Cleanup
cDNA amplification
Dynabeads
cDNA quantification
SenNet
TriState
snRNAseq
Lung
PCLS
Frozen tissue
Abstract
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)
Attachments
Steps
Dynabeads
Add 125µL 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).
Slowly remove and discard 125µLRecovery Agent/Partitioning Oil (pink) from the bottom of the tube.
Prepare Dynabeads Cleanup Mix:
| A | B | C | D |
|---|---|---|---|
| Reagents | 1X (μl) | 4X+10% (μl) | 8X+10% (μl) |
| Cleanup Buffer | 182 | 801 | 1602 |
| Dynabeads MyOne SILANE | 8 | 35 | 70 |
| Reducing Agent B | 5 | 22 | 44 |
| Nuclease-free Water | 5 | 22 | 44 |
| Total | 200 | 880 | 1760 |
Calculations for Dynabeads Cleanup Mix preparation.
Vortex and add 200µL to each sample. Pipette mix 10x (pipette set to 200 µl).
Incubate 0h 10m 0s at Room temperature .
Pipette mix again at ~0h 5m 0s after start of incubation to resuspend settled beads.
Prepare Elution Solution I. Vortex and centrifuge briefly.
Elution Solution I
| A | B | C |
|---|---|---|
| Reagents | 1X (μl) | 10X (μl) |
| Buffer EB | 98 | 980 |
| 10% Tween 20 | 1 | 10 |
| Reducing Agent B | 1 | 10 |
| Total | 100 | 1000 |
Calculations for Elution Solution I preparation.
At the end of 0h 10m 0s incubation, place on a 10x Magnetic Separator. High position (magnet.High) until the solution clears.
Remove the supernatant (aqueous phase and Recovery Agent).
Add 300µL 80% ethanol to the pellet while on the magnet. Wait 0h 0m 30s.
Remove the ethanol.
Add 200µL 80% ethanol to pellet. Wait 0h 0m 30s.
Remove the ethanol.
Centrifuge briefly. Place on the magnet. Low.
Remove remaining ethanol. Air dry for 0h 1m 0s.
Remove from the magnet. Immediately add 35.5µL Elution Solution I.
Pipette mix (pipette set to 30 µl) without introducing bubbles.
Incubate 0h 2m 0s at Room temperature.
Place on the magnet. Low until the solution clears.
Transfer 35µL sample to a new tube strip.
cDNA amplification
Prepare cDNA Amplification Mix On ice. Add reagents in the order listed. Vortex and centrifuge briefly.
cDNA Amplification Reaction Mix
| A | B | C | D |
|---|---|---|---|
| Reagents | 1X (μl) | 4X+10% (μl) | 8X+10% (μl) |
| Amp Mix | 50 | 220 | 440 |
| cDNA Primers | 15 | 66 | 132 |
| Total | 65 | 286 | 572 |
Calculations for cDNA Amplification Reaction Mix preparation.
Add 65µL cDNA Amplification Reaction Mix to 35µL sample.
Pipette mix 15x (pipette set to 90 µl). Centrifuge briefly.
Incubate in a thermal cycler with the following protocol:
| A | B | C |
|---|---|---|
| Lid Temperature | Reaction Volume | Run Time |
| 105°C | 100 µl | ~30-45 min |
| Step | Temperature | Time |
| 1 | 98°C | 3 min |
| 2 | 98°C | 15 sec |
| 3 | 63°C | 20 sec |
| 4 | 72°C | 1 min |
| 5 | Go to Step 2 – 11 cycles | |
| 6 | 72°C | 1 min |
| 7 | 4°C | Hold |
Thermocycler protocol.
Store at 4°C for up to 72h 0m 0s or -20°C for ≤1 week or proceed to the next step.
cDNA Cleanup – SPRIselect:
Vortex to resuspend the SPRIselect reagent. Add 60µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 150 µl).
Incubate 0h 5m 0s at Room temperature.
Place on the magnet. High until the solution clears.
Remove the supernatant.
Add 200µL 80% ethanol to the pellet. Wait 0h 0m 30s.
Remove the ethanol.
Repeat steps 29 and 30 for a total of 2 washes.
Centrifuge briefly and place on the magnet. Low.
Remove any remaining ethanol. Air dry for 0h 2m 0s.
Remove from magnet. Add 40.5µL Buffer EB. Pipette mix 15x.
Incubate for 0h 2m 0s at Room temperature.
Place the tube strip on the magnet. High until the solution clears.
Transfer 40µL sample to a new tube strip.
Store at 4°C for up to 72h 0m 0s or at -20°C for up to 4 weeks, or proceed to the next step.
cDNA Quantification
Run 1µL sample (Dilution Factor 1:10) on an Agilent Bioanalyzer High Sensitivity chip.
