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Blue:RNA-Seq Experiments:11072016
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== Experiment == *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=== {| {{table}} | align="center" style="background:#f0f0f0;"|'''Sample''' | align="center" style="background:#f0f0f0;"|'''Total Reads''' | align="center" style="background:#f0f0f0;"|'''% Genome''' | align="center" style="background:#f0f0f0;"|'''% ERCC''' | align="center" style="background:#f0f0f0;"|'''% Unmapped''' | align="center" style="background:#f0f0f0;"|'''% Unique''' | align="center" style="background:#f0f0f0;"|'''% 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.'''
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