Blue:RNA-Seq Experiments:11072016: Difference between revisions

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>B1lake
(Created page with "== 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 n...")
 
>B1lake
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*Procedure:
*Procedure (as per tube control protocol for V4):
 
"""Lysis Buffer (no PolydIdC):"""
 
#C1 loading reagent + ERCC | 0.5ul
#3' Smart-seq CDS primer IIA | 1.2ul
#10x Reaction buffer | 1.3ul
Nuclease Free Water | 5ul
 
*Combine:
#Lysis Mix | 2ul
#Triton X-100 +/- PolydIdC | 0.5ul
 
*TritonX100 PolydIdC mixes:
#0.5% Triton X-100:
##1ul 10% + 19ul dH2O
 
#1% Triton X-100:
##0.5ul 10% + 19ul dH2O
 
#0.5% Triton X-100 + PolydIdC:
##1ul 10% + 13.9ul PolydIdC + 5.1ul dH2O
 
#1% Triton X-100 + PolydIdC:
##0.5ul 10% + 13.9ul PolydIdC + 5.6ul dH2O

Revision as of 18:17, 7 November 2016

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 + PolydidC
  5. 1x Lysis + 0.1% Triton X-100 + PolydidC
  6. 1x Lysis + 0.2% Triton X-100 + PolydidC
  7. 1x Lysis + vortex 30s
  8. NTC


  • Procedure (as per tube control protocol for V4):

"""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

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. 0.5ul 10% + 19ul dH2O
  1. 0.5% Triton X-100 + PolydIdC:
    1. 1ul 10% + 13.9ul PolydIdC + 5.1ul dH2O
  1. 1% Triton X-100 + PolydIdC:
    1. 0.5ul 10% + 13.9ul PolydIdC + 5.6ul dH2O