Arichard:Protocols/CEL Seq

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Revision as of 17:33, 21 February 2013 by >Andrew
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CEL-Seq protocol validation as of 2/21/2013

  • 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

  • RT primers must be diluted to appropriate concentrations depending on the input:
    • For 1 ng of total RNA, use 0.5 ul of 32 pmol/ul.
    • For 100 pg of total RNA, use 0.5 ul of 3.2 pmol/ul.
    • For 10 pg of total RNA, use 0.5 ul of 0.32 pmol/ul.
  • See diluted RT primer layout under Arichard:Reagents.

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 @ 42 degC for RT rxn.

Second strand synthesis

  1. Move tubes to ice block and add 8 ul of second strand mix per rxn:
    1. 6.3 ul DDW H2O.
    2. 1 ul Second Strand buffer.
    3. 0.4 ul dNTP mix.
    4. 0.2 ul DNA Polymerase
    5. 0.1 ul RNaseH.
  2. Flick and spin to collect, then incubate 2 hrs @ 16 degC.
  3. Pool samples and add to UV treated EtOH precipitation mix (for 16 samples, 10 ul/samples):
    1. 400 ul 100% EtOH.
    2. 32 ul 3M NaAc.
    3. 1 ul glycoblue.
  4. Incubate at least 1 hr @ -80 degC.
  5. Spin 30 min @ 4 degC.
  6. Remove supernatant and wash with 750 ul 75% EtOH.
  7. Spin again 10 min @ 4 degC.
  8. Remove supernatant and let dry 5 min.
  9. Resuspend in 4.5 ul UV treated H2O.
  • Stopping point: Store @ -20 degC.

Common primer

  1. Add 0.5 ul of 100 pmol/ul CS-5primeAD-T7 primer to 4.5 ul of ds cDNA.
  2. Add 5 ul of 2X Phusion Flash mix and incubate 30 min @ 72 degC.

IVT

  1. On ice, add 15 ul of IVT mix per rxn:
    1. 2.5 ul ATP.
    2. 2.5 ul CTP.
    3. 2.5 ul GTP.
    4. 2.5 ul UTP.
    5. 2.5 ul 10X T7 buffer.
    6. 2.5 ul T7 enzyme.
  2. Flick and spin to collect. Incubate 13 hrs @ 37 degC, followed by 4 degC forever.

Mg2+ fragmentation

  1. On ice, add 6.25 of 10X NEB fragmentation buffer to 25 ul of sample.
  2. Incubate 3 min @ 94 degC.
  3. Immediately move to ice and add 2 ul fragmentation stop buffer.