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