Arichard:Protocols/CEL Seq: Difference between revisions

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>Andrew
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## Add 1 ul 100 ng/ul RNA to 9 ul H2O --> 10 ul 10 ng/ul RNA.
## Add 1 ul 100 ng/ul RNA to 9 ul H2O --> 10 ul 10 ng/ul RNA.
## Add 2 ul 10 ng/ul RNA to 8 ul of H2O --> 10 ul 2 ng/ul RNA.
## Add 2 ul 10 ng/ul RNA to 8 ul of H2O --> 10 ul 2 ng/ul RNA.
# Dilute ERCC RNA Spike-in Control to 1:10,000, on ice:
## Add 1 ul of undiluted ERCC to 9 ul H2O --> 10 ul 1:10 ERCC.
## Add 1 ul of 1:10 ERCC to 9 ul H2O --> 10 ul 1:100 ERCC.
## Add 1 ul of 1:100 ERCC to 9 ul H2O --> 10 ul 1:1000 ERCC.
## Add 1 ul of 1:1000 ERCC to 9 ul H2O --> 10 ul 1:10000 ERCC.
# Add 5 ul 2 ng/ul RNA to 2 ul 1:10000 ERCC --> 7 ul 1.42 ng/ul RNA 1:35000 ERCC.
## Add 0.7 ul of this to 0.5 ul 32 pmol/ul primer for 1 ng total RNA samples.
## Need total of 2 x 0.7 ul + 2 ul for dilution to 142 pg/ul RNA 1:350000 ERCC = 3.4 ul.
# Add 2 ul 1.42 ng/ul RNA 1:35000 ERCC to 18 ul H2O --> 20 ul 142 pg/ul RNA 1:350000 ERCC.
## Add 0.7 ul of this to 0.5 ul 3.2 pmol/ul primer for 100 pg total RNA samples.
## Need 14 x 0.7 ul + 1 ul for dilution to 14.2 pg/ul RNA 1:3500000 ERCC = 10.8 ul.
# Add 1 ul 142 pg/ul RNA 1:350000 ERCC to 9 ul H2O --> 10 ul 14.2 pg/ul RNA 1:3500000 ERCC.
## Add 0.7 ul of this to 0.5 ul 0.32 pmol/ul primer for 10 pg samples.
## Need 12 x 0.7 ul = 8.4 ul.

Revision as of 00:52, 8 February 2013

CEL-Seq protocol validation

  • Protocol modified to use common sequence primer with Illumina 5' Adapter and T7 promoter (CS-5primeAD-T7).
  • 100 ng/ul total RNA will be used as input (from Rui).
  • I will work with 16 tubes at a time.
  • For one set of 16 tubes, I want 2 x 1 ng rxns (+ control), 12 x 100 pg rxns (experimental), and 2 x no RNA (no template controls, NTC).
  • For the second set of 16, I want 2 x 100 pg rxns (+ control), 12 x 10 pg rxns (experimental, approx. single cell amount), and 2 x no RNA (NTCs).
  • Keep all RNA on ice at all times.
  • Work in the hood. EtOH, RNase away, and UV treat all surfaces and tools. UV treat water for dilutions.
  • Use low-bind tubes.

Prep

  • Sample layout is as follows:
Sample # RNA input Sample # RNA input Sample # RNA input Sample # RNA input
1 1 ng 9 100 pg 17 100 pg 25 10 pg
2 1 ng 10 100 pg 18 100 pg 26 10 pg
3 100 pg 11 100 pg 19 10 pg 27 10 pg
4 100 pg 12 100 pg 20 10 pg 28 10 pg
5 100 pg 13 100 pg 21 10 pg 29 10 pg
6 100 pg 14 100 pg 22 10 pg 30 10 pg
7 100 pg 15 NTC 23 10 pg 31 NTC
8 100 pg 16 NTC 24 10 pg 32 NTC
  • Barcoded RT primers are in 48 wells of a 92 well plate at 200 uM each, in the following layout:
Barcode number 1 2 3 4 5 6 7 8 9 10 11 12
A 1 2 3 4 5 6 7 8 9 10 11 12
B 13 14 15 16 17 18 19 20 21 22 23 24
C 25 26 27 28 29 30 31 32 33 34 35 36
D 37 38 39 40 41 42 43 44 45 46 47 48
E Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty
F Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty
G Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty
H Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty
  • Serial dilutions will be necessary to dilute the RT primers to the correct concentration.

Dilute RT primers to appropriate concentrations

  1. Dilute primers to 32 pmol/ul, 3.2 pmol/ul, and 0.32 pmol/ul, depending on input (Note that each primer must be diluted separately for each sample):
    1. Add 16 ul of 100 pmol/ul primer to 84 ul H2O --> 100 ul 32 pmol/ul primer. Use 0.5 ul of this per 1 ng total RNA.
    2. Add 10 ul of 32 pmol/ul primer to 90 ul H2O --> 100 ul 3.2 pmol/ul primer. Use 0.5 ul of this per 100 pg total RNA.
    3. Add 10 ul of 3.2 pmol/ul primer to 90 ul H2O --> 100 ul 0.32 pmol/ul primer. Use 0.5 ul of this per 10 pg total RNA.
  2. Dilute total RNA to 2 ng/ul, on ice:
    1. Add 1 ul 100 ng/ul RNA to 9 ul H2O --> 10 ul 10 ng/ul RNA.
    2. Add 2 ul 10 ng/ul RNA to 8 ul of H2O --> 10 ul 2 ng/ul RNA.
  3. Dilute ERCC RNA Spike-in Control to 1:10,000, on ice:
    1. Add 1 ul of undiluted ERCC to 9 ul H2O --> 10 ul 1:10 ERCC.
    2. Add 1 ul of 1:10 ERCC to 9 ul H2O --> 10 ul 1:100 ERCC.
    3. Add 1 ul of 1:100 ERCC to 9 ul H2O --> 10 ul 1:1000 ERCC.
    4. Add 1 ul of 1:1000 ERCC to 9 ul H2O --> 10 ul 1:10000 ERCC.
  4. Add 5 ul 2 ng/ul RNA to 2 ul 1:10000 ERCC --> 7 ul 1.42 ng/ul RNA 1:35000 ERCC.
    1. Add 0.7 ul of this to 0.5 ul 32 pmol/ul primer for 1 ng total RNA samples.
    2. Need total of 2 x 0.7 ul + 2 ul for dilution to 142 pg/ul RNA 1:350000 ERCC = 3.4 ul.
  5. Add 2 ul 1.42 ng/ul RNA 1:35000 ERCC to 18 ul H2O --> 20 ul 142 pg/ul RNA 1:350000 ERCC.
    1. Add 0.7 ul of this to 0.5 ul 3.2 pmol/ul primer for 100 pg total RNA samples.
    2. Need 14 x 0.7 ul + 1 ul for dilution to 14.2 pg/ul RNA 1:3500000 ERCC = 10.8 ul.
  6. Add 1 ul 142 pg/ul RNA 1:350000 ERCC to 9 ul H2O --> 10 ul 14.2 pg/ul RNA 1:3500000 ERCC.
    1. Add 0.7 ul of this to 0.5 ul 0.32 pmol/ul primer for 10 pg samples.
    2. Need 12 x 0.7 ul = 8.4 ul.