GSM8950737: CTC (LM2) - 5542-8; Homo sapiens; RNA-Seq
Single cells were visualized in chamber slides coated with polyHEMA for isolation and deposited in tubes via Rarecyte aspirating needle in 400-600nL PBS. Individual tubes containing deposited cells were processed using SMARTseq2 protocol as previously described23 with several modifications. Shortly, 2uL of lysis buffer was added to each tube, consisting of Triton, RNase inhibitor, dNTPs, and Oligo dT, and incubated first at room temp for 5min, then at 72°C for 5min. Reverse transcription mix was added (3uL), containing 5x first strand buffer, RNase inhibitor, DTT, MgCl2, Betaine, TSO, and Smart scribe reagent. Samples were incubated at 42°C for 90min, then at 70°C for 5min. Amplification cycle number was determined for each set of cells using iTaq SYBR green reagent and run for 40 cycles. Amplification mix, containing IS PCR primer, KAPA HiFi hotstart, and Lambda exonuclease, was added (7.5uL) and amplification was run at ~28-35 cycles depending on the single cell. Libraries were prepared for sequencing from amplified cDNA using tagmentation via Nextera XT library prep kit (library kit FC-131-1096, index kit FC-131-1001) and standard kit protocol. Libraries were pooled and sequenced on a NextSeq550 platform. Data was demultiplexed using Basespace BCL Convert v1.2.1.