title: GSM8505855: SUM159_EGFP+_dsRed+; Homo sapiens; RNA-Seq
library_construction_protocol: Xenograft tumors were harvested and digested using 5mL digestion media (phenol red-free DMEM/F12 [Corning cat. 16-405-CV], 100mg/mL gentamicin [Worthington, cat. LS004176], 250U/mL collagenase II [Worthington cat. LS004174] and 2mg/mL DNase I [Stemcell, cat. 07900]) per 1g tumor at 37oC in a gentleMACS dissociator (Miltenyi). The tumor digestion was passed through a 200mM filter and centrifuged at 300g for 1 minute. Red blood cells were lysed by adding 9mL ddH2O to the cell pellet for 20 seconds and lysis was stopped by the addition of 1mL 10X PBS. To dissociate cell clumps further, the cell suspension was centrifuged at 300 g for 1 minute, then resuspended in TrypLE Express (Gibco, cat. 12605-010) for 5 minutes. To stop the digestion, 9mL of PBS was added, and the cell suspension was passed through a 40mm cell strainer. SUM159, BCM-4272, and BCM-15006 were sorted based on fluorescent reporter activity (SUM159: EGFP+/mNeptune2+, EGFP+/dsRed+, EGFP-/mNeptune2+, EGFP-/dsRed+; BCM-4272 and BCM-15006: EGFP+ or EGFP-). Single-cell gene expression libraries were prepared according to Chromium Single Cell Gene Expression 3v3.1 kit (10x Genomics). In brief, single cells, reverse transcription (RT) reagents, Gel Beads containing barcoded oligonucleotides, and oil were loaded on a Chromium controller (10x Genomics) to generate single-cell GEMS (Gel Beads-In-Emulsions) where full-length cDNA was synthesized and barcoded for each single cell. Subsequently the GEMS are broken and cDNA from each single cell are pooled. Following cleanup using Dynabeads MyOne Silane Beads, cDNA is amplified by PCR. The amplified product is fragmented to optimal size before end-repair, A-tailing, and adaptor ligation. The final library was generated by amplification.