Arichard:Protocols/CEL Seq: Difference between revisions

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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.
* See diluted RT primer layout.


====Dilute total RNA to 2 ng/ul, on ice====
====Dilute total RNA to 2 ng/ul, on ice====
# 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.
* Store all at -80 degC.


====Dilute ERCC RNA Spike-in Control to 1:10,000, on ice====
====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 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 1:10 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 1:100 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 1 ul of 1:1000 ERCC to 9 ul H2O --> 10 ul 1:10000 ERCC.
* Store all at -80 degC.


===Protocol===
====Mix total RNA and ERCC RNA Spike-in Control====
 
====RT reaction, first strand====


# Add 5 ul 2 ng/ul RNA to 2 ul 1:10000 ERCC --> 7 ul 1.42 ng/ul RNA 1:35000 ERCC.
# Add 5 ul 2 ng/ul RNA to 2 ul 1:10000 ERCC --> 7 ul 1.42 ng/ul RNA 1:35000 ERCC.
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## Need 12 x 0.7 ul = 8.4 ul.
## Need 12 x 0.7 ul = 8.4 ul.


to be continued
===Protocol===
 
====RT reaction, first strand====
 
# On ice block, add 0.5 ul of each RT primer at appropriate concentration (see prep above) to each tube.
# Add 0.7 ul of each sample/ERCC mixture to respective tubes.
# Flick and spin tubes to collect, then return to ice block.
# Incubate 10 min at 70 degC to anneal RT primers.
# Add 0.8 ul of RT mix per rxn:
## 0.2 ul 10X first strand buffer.
## 0.4 ul dNTP mix.
## 0.1 ul RNase inhibitor.
## 0.1 ul ArrayScript.
# Flick and spin tubes to collect, then return to ice block.
# Incubate 2 hrs at 42 degC for RT rxn.
# Move tubes to ice block and add 8 ul of second strand mix per rxn:
##

Revision as of 20:37, 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.
  • See diluted RT primer layout.

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.
  • Store all at -80 degC.

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.
  • Store all at -80 degC.

Mix total RNA and ERCC RNA Spike-in Control

  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.

Protocol

RT reaction, first strand

  1. On ice block, add 0.5 ul of each RT primer at appropriate concentration (see prep above) to each tube.
  2. Add 0.7 ul of each sample/ERCC mixture to respective tubes.
  3. Flick and spin tubes to collect, then return to ice block.
  4. Incubate 10 min at 70 degC to anneal RT primers.
  5. Add 0.8 ul of RT mix per rxn:
    1. 0.2 ul 10X first strand buffer.
    2. 0.4 ul dNTP mix.
    3. 0.1 ul RNase inhibitor.
    4. 0.1 ul ArrayScript.
  6. Flick and spin tubes to collect, then return to ice block.
  7. Incubate 2 hrs at 42 degC for RT rxn.
  8. Move tubes to ice block and add 8 ul of second strand mix per rxn: