GSM7898950: persister_D2-2, replicate 2, scRNA-seq; Homo sapiens; RNA-Seq
Tumors were minced with a razor blade and enzymatically dissociated in two steps. First, minced samples were incubated with 5 mL of digestion mix 1 (9 mL Advanced DMEM/F12, 1 mL Gentle Collagenase and 100 μL DNase I) at 37°C for 30 min, centrifuged at 1000 RPM for 4 min. After removing the supernatant, samples were incubated with 5 mL of digestion mix 2 (3 mL Advanced DMEM/F12, 2 mL Dispase, 5 mL TrypLE, 100 μL DNase I) at 37°C for 15 min, placed on a 70 μm cell strainer and washed with Advanced DMEM/F12 + 10% FBS. Samples were centrifuged at 1000 RPM for 4 min, incubated with 1 ml of ACK Lysing Buffer (ThermoFisher) for 4 min, washed with PBS + 10% FBS, resuspended in PBS, placed on a 35 μm cell strainer and viability was determined by Trypan Blue staining. Dissociated cells were collected for scRNA-Seq immediately (resistance experiment) or cryopreserved (dose-response experiment) by resuspending in freezing media (50% FBS, 40% Advanced DMEM/F12 and 10% DMSO) and stored at -80°C. Batches of replicates were processed on the same day. Frozen samples were thawed, washed in media, resuspended in PBS and the cell concentration adjusted for the 10X Genomics single-cell encapsulation protocol following library preparation and sequencing. Library preparation was performed using the 10X Genomics Chromium platform with the Chromium Single Cell Library & Gel Bead Kit v3.1 following the manufacturer's instructions.