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