GSM5412064: single-cell-derived clonal populations, collected 24h after  treatment with Gefitinib drug; Homo sapiens; RNA-Seq
Lysis of PC-9 clonal populations prior to transcriptome analysis: In order to process each clonal population to transcriptome analysis by MARS-seq, clones were washed with 150 µL PBS (Biological Industries, 02-023-1A) and detached by the addition of 30 µL trypsin b (Biological Industries, #03-052-1B) for 3 minutes at 37°C. 1um of Lysis buffer per 50cells was added. The lysis buffer contained 0.005% RNAse-Inhibitor (RNAsin plus, Promega, N2611) and 10% Triton X-100 (Sigma Aldrich, 9002-93-1). Preparaion of bulk cDNA library by MARS-seq: In a similar assay to Meir et al. 2020, lysed clonal populations were manually transferred in four replicates of 1µL into MARS-Seq 384-well plates containing 2µL of barcoded RT primers (concentration of 8 nM in each well). Downstream library preparation was done according to Jaitin et al. 2014, and by using a randomized UMI sequence of 8 base-pairs (allowing a maximal count of ~65K UMIs per gene per well). Library preparation was identical to that used for single cells (Jaitin et al. 2014), with the only exception of extending the 95°C evaporation step before RT1 from 3 minutes to 4 minutes, in order to compensate for the higher volume.