Chromium RNA v3.1
1. GEM Generation & Barcoding
Getting Started !
Equilibrate to room temperature before use:
- Single Cell 3' Gel Beads (-80°C)
- RT Reagent B
- Template Switch Oligo (-80°C)
- Reducing Agent B
Template Switch Oligo: Centrifuge briefly, resuspend in 80 µl Low TE Buffer. Vortex 15 sec at maximum speed, centrifuge briefly, leave at room temperature for ≥ 30 min. After resuspension, store at -80°C.
Obtain:
- 50% glycerol solution (if <8 samples)( 1 tube/ 2 channels)
- Partitioning Oil (300 µl/ sample)
- Chromium Next GEM Chip G
- 10x Chip Holder
- 10x Gasket
Place on ice:
- RT Enzyme C
- Chilled Metal Block
1.1 Preparing Master Mix ON ICE
- add reagents in order as shown
Master Mix 1 2 3 4 5 6 7 8 RT Reagent B 18.80 39.48 59.22 78.96 98.70 118.44 138.18 157.92 Template Switch Oligo 2.40 5.04 7.56 10.08 12.60 15.12 17.64 20.16 Reducing Agent B 2.00 4.20 6.30 8.40 10.50 12.60 14.70 16.80 RT Enzyme C 8.70 18.27 27.41 36.54 45.68 54.81 63.95 73.08 Total 31.80 66.78 100.17 133.56 166.95 200.34 233.73 267.12
- Pipette mix 15x, centrifuge briefly - add 31,8 µl Master Mix into each o a PCR 8-tube strip on ice.
If you get a sample with a volume of 10 or 5 µl add water to the Master Mix: Volume of Sample 1 2 3 4 5 6 7 8 10 µl (43,2 µl - 10 µl = 33,2 µl) 33.2 69.72 104.58 139.44 174.3 209.16 244.02 278.88 5 µl (43,2 µl - 5 µl = 38,2 µl) 38.2 80.22 120.33 160.44 200.55 240.66 280.77 320.88