GSM8224528: Replicate 2; Mus musculus; RNA-Seq
1ul harsh lysis buffer (50mM Tris (pH=8.0), 5mM EDTA (pH=8.0), 10mM DTT, 1% Tween-20, 1% Triton X-100, 0.1g/l proteinase K, 2.5mM dNTPs, and ERCC Mix (107-fold dilution) and 1ul 10mM barcoded RT primer was added to each well. Plates were incubated at 50°C for 5 mins to lyse cells and proteinase K was heat-inactivated by subsequently incubating at 80°C for 20 mins. To minimize contamination across wells, heavy-duty plate seals and qPCR compression pads (Thermo Fisher Scientific, Cat. 4312639) were used to seal the plates. The lysis reaction was mixed with 2ul reverse transcription master mix 5×buffer (Thermo Fisher Scientific, Cat. 11756500), 2ul 5M Betaine (Sigma-Aldrich, Cat. B0300-1VL), 0.2ul 50mM E5V6NEXT template switch oligo (Integrated DNA Technologies), 0.1ul 200U/ul Maxima H-RT (Thermo Fisher Scientific, Cat. EP0751), 0.1ul 40U/ul NxGen RNase Inhibitor, and 0.6ul nuclease-free water (Thermo Fisher Scientific, Cat. AM9932). The reaction system was then incubated at 42°C for 1.5 hrs, followed by 10 mins at 75°C to inactivate reverse transcriptase. PCR was performed by adding 10ul 2×HiFi PCR mix (Kapa Biosystems, Cat. 7958927001) and 0.5ul 60mM SINGV6 primer and running the following program: 98°C for 3 mins, 20 cycles of 98°C for 20 s, 64°C for 15 s, 72°C for 4 mins, with a final extension step off 5 mins at 72°C. Single-cell cDNA was pooled by plate to make library. 600 pg cDNA from each sample plate was used in a modified Nextera XT (Illumina, Cat. FC-131-1024) library preparation but using the P5NEXTPT5 primer and the tagmentation time of 5 mins. The resulting libraries were purified following the Nextera XT protocol (0.6× ratio) and quantified by qPCR using Kapa Library Quantification kit (Kapa Biosystems, Cat. KK4824).