Blue:RNA-Seq Experiments:11072016

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Revision as of 20:29, 7 November 2016 by >B1lake (→‎Experiment)
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Overview

  • Test Smarter v4 chemistry lysis conditions for nuclei
    • Initial C1 runs on v4 chemistry showed lower genome mapping rates, therefore it is possible that the new lysis conditions (i.e. 1x lysis buffer) are not optimal for nuclei
    • The HT C1 chips run v3 chemistry and have the same lysis conditions, therefore we want to determine whether we can optimize lysis for nuclei
  • Test whether polydIdC has any beneficial effect on the new V4 chemistry


Experiment

  • Tube control (100 nuclei) test conditions:
  1. 1x Lysis
  2. 1x Lysis + 0.1% Triton X-100
  3. 1x Lysis + 0.2% Triton X-100
  4. 1x Lysis + 0.1% Triton X-100 + PolydidC
  5. 1x Lysis + vortex 30s
  6. NTC
  • note: to mimic C1 conditions, lysis buffer will be added without mixing to cells unless stated



Procedure (as per tube control protocol for V4):
Modifications to Lysis Buffer (no PolydIdC):
  1. C1 loading reagent + ERCC | 0.5ul
  2. 3' Smart-seq CDS primer IIA | 1.2ul
  3. 10x Reaction buffer | 1.3ul
  4. Nuclease Free Water | 5ul


Combine:
  1. Lysis Mix | 2ul
  2. Triton X-100 +/- PolydIdC | 0.5ul


TritonX100 PolydIdC mixes:
  1. 0.5% Triton X-100:
    1. 1ul 10% + 19ul dH2O
  1. 1% Triton X-100:
    1. 2ul 10% + 18ul dH2O
  1. 0.5% Triton X-100 + PolydIdC:
    1. 1ul 10% + 13.9ul PolydIdC + 5.1ul dH2O