Arichard:Protocols/CEL Seq

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Revision as of 00:26, 8 February 2013 by >Andrew (Created page with "==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 use...")
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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).
  • 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

Prep

Dilute primers to appropriate concentrations

  1. Resuspend 100 nmol barcode RT primers in 100 ul H2O --> 100 ul 1000 pmol/ul.