Arichard:Protocols/CEL Seq
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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
- Resuspend 100 nmol barcode RT primers in 100 ul H2O --> 100 ul 1000 pmol/ul.