GSM6503161: HEK293T, ERCC spike-in mix, pooling of 10 indexed cells, LAST-seq; Homo sapiens; RNA-Seq
Single cells were flow-sorted into 96-well plates with 2 µl lysis buffer in each well, containing 0.1% Triton X-100, 2 nM LAST-seq primer, 0.1 U/µl Rnase Inhibitor, 0.5 mM dNTP, 60 pg RNA carriers, and the RNA spike-in. The plate was then sealed with aluminum films (MilliporeSigma), briefly spun down, and lysed at 65°C for 5 min before keeping on ice. Single cells were sorted into 96-well plates containing lysis buffer, followed by 65°C heating and 30min vacuum evaporation.  RNase H and DNA pol mixture were added to each well, followed by quick shaking, centrifuge, and 30min incubation at 37°C. The reaction buffer in each well was pooled and purified by RNA cleanup beads. The beads were rehydrated by IVT reaction buffer and incubated at 37°C up to 16 hrs. The resultant product was purified by RNA cleanup beads and was converted to cDNA by reverse transcription. Then, the cDNA was amplified by primers containing P5/P7 adaptors and purified by DNA cleanup beads. The resultant product was enriched by PCR and subjected to illumina sequencing. LAST-seq, Cel-seq2 and SMART-seq V4 kit