Arichard:Protocols/CEL Seq: Difference between revisions

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===Prep===
===Prep===
* Sample layout is as follows:
{| {{table}}
| align="center" style="background:#f0f0f0;"|'''Sample #'''
| align="center" style="background:#f0f0f0;"|'''RNA input'''
| align="center" style="background:#f0f0f0;"|'''Sample #'''
| align="center" style="background:#f0f0f0;"|'''RNA input'''
| align="center" style="background:#f0f0f0;"|'''Sample #'''
| align="center" style="background:#f0f0f0;"|'''RNA input'''
| align="center" style="background:#f0f0f0;"|'''Sample #'''
| align="center" style="background:#f0f0f0;"|'''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
|-
|
|}


====Dilute RT primers to appropriate concentrations====
====Dilute RT primers to appropriate concentrations====
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## 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.
## 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.
## 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.
## 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.


====Dilute total RNA to 2 ng/ul, on ice====
====Dilute total RNA to 2 ng/ul, on ice====

Revision as of 17:20, 14 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

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.

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.

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.

Protocol

RT reaction, first strand

  1. 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.
  2. 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.
  3. 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.

to be continued