Blue:RNA-Seq Experiments:11072016

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Overview[edit]

  • 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[edit]

  • Nuclei used:
    • 5340 Frontal Cortex (09-19-2016)


  • 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 (PolydIdC lysis)
  • 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:

  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 base lysis buffer with TritonX-100 +/- PolydIdC mixes:

  1. Lysis Mix | 2ul
  2. Mixes | 0.5ul


TritonX100 +/- PolydIdC mixes:

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



Check cDNA yields

  • Added 10ul water to PCR products
  • Used 1ul for Qubit quantification:
  1. 1x Lysis - 3.10ng/ul
  2. 1x Lysis + 0.1% Triton X-100 - 3.27ng/ul
  3. 1x Lysis + 0.2% Triton X-100 - 3.68ng/ul
  4. 1x Lysis + 0.1% Triton X-100 + PolydidC - 21.0ng/ul
  5. 1x Lysis + vortex 30s - 3.64ng/ul
  6. NTC (PolydIdC lysis) - 22.8ng/ul
  7. NTC (PCR Mix only) - 0.18ng/ul

Sequencing Outcome[edit]

Sample Total Reads % Genome % ERCC % Unmapped % Unique % Multiple
1x Lysis 122 29.72972973 70.27027027 8.196721311 90.98360656 0.819672131
1x Lysis + 0.1% Triton X-100 8563187 23.16858324 76.83141676 5.019778267 94.23211241 0.74810932
1x Lysis + 0.2% Triton X-100 76650 38.53267571 61.46732429 10.00130463 88.87671233 1.12198304
1x Lysis + 0.1% Triton X-100 + PolydidC 1670521 38.61607897 61.38392103 10.36724471 88.54566929 1.087086005
1x Lysis + vortex 30s 14298673 36.81574225 63.18425775 6.449094961 92.65633251 0.894572524
NTC (PolydIdC lysis) 829421 3.098196578 96.90180342 13.85930667 85.90076692 0.239926406


Outcome: Looks like 0.1% Triton-X supplement and PolydIdC can bring up the genome mapping rate to that of the Lysis+vortex control condition while also significantly increasing cDNA yields.