Human K562 cells (ATCC) were grown in RPMI medium supplemented with 10% heat-inactivated FBS, 2 mM l-glutamine, 0.1 mM 2-mercaptoethanol, 100 units/ml penicilin G sodium, and 100 μg/ml streptomycin sulfate. NIH3T3 mouse fibroblasts (ATCC) were cultured in DMEM media containing 10% bovine calf serum, 4 mM l-glutamine and 100 units/ml penicillin G sodium and 100 μg/ml streptomycin sulfate. For both K562 and NIH3T3, single cell suspensions of 100 000 cells were prepared and lysed in 500 μl volume either using freeze-thaw cycles (in freeze-thaw lysis buffer; 100 mM Tris, pH 7.5, 10 mM EDTA, 1 M NaCl, 5 μM DTT, 0.4 U/ml Lucigen RNase) or detergent-based buffer as used in DropSeq method (17 (link)) (100 mM Tris, pH 7.5, 10 mM EDTA, 3% Ficoll PM-400, 0.1% Sarkosyl, 25 mM DTT). For freeze-thaw lysis, lysates were prepared for one, two and three freeze-thaw cycles for comparison. Following lysis, mRNA from lysates were isolated using magnetic oligo-dt beads (NEB, S1419S) following manufacturer's instructions. mRNA was quantified using NanoDrop 2000 spectrophotometer. For qPCR, 10 ng of mRNA was used as input for all conditions and reactions were run on CFX Connect Real-Time PCR machine (Biorad) using SsoFast EvaGreen PCR supermix (Biorad) following manufacturer's instructions. Primers were obtained from Sigma (KiCqStart primers).