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:
- 1x Lysis
- 1x Lysis + 0.1% Triton X-100
- 1x Lysis + 0.2% Triton X-100
- 1x Lysis + 0.1% Triton X-100 + PolydidC
- 1x Lysis + vortex 30s
- 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:
- C1 loading reagent + ERCC | 0.5ul
- 3' Smart-seq CDS primer IIA | 1.2ul
- 10x Reaction buffer | 1.3ul
- Nuclease Free Water | 5ul
Combine base lysis buffer with TritonX-100 +/- PolydIdC mixes:
- Lysis Mix | 2ul
- 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:
- 1x Lysis - 3.10ng/ul
- 1x Lysis + 0.1% Triton X-100 - 3.27ng/ul
- 1x Lysis + 0.2% Triton X-100 - 3.68ng/ul
- 1x Lysis + 0.1% Triton X-100 + PolydidC - 21.0ng/ul
- 1x Lysis + vortex 30s - 3.64ng/ul
- NTC (PolydIdC lysis) - 22.8ng/ul
- 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.