GSM8230917: Simurosertib; Homo sapiens; RNA-Seq
Upon delivery to the lab, samples were mechanically/enzymatically dissociated using the tumor dissociation kit (#130-095-929, Miltenyi) and the GentleMACS Octo Dissociator with Heaters (Miltenyi, # 130-096-427). Resection samples were chopped and added to 7.5 mL of enzyme mix in the GentleMACS tube, while core needle biopsies/fine needle aspiration samples were added to 2.5 mL of enzyme mix in the GentleMACS tube. After 15-30 minutes dissociation, depending on sample size and consistency, bigger size samples were filtered with MACS SmartStrainers (70 μm) (Miltenyi, #130-098-462) into 50 mL tubes, and smaller samples were filtered with 35 uM stainer cap FACS tube (Corning # 352235). Then, samples were centrifuged (800g, 1 minute) and supernatant was discarded. Pelleted cells were then stained as indicated below. Cell pellet was resuspended in 200-3000 uL of Red Blood Cell Lysis Solution (ACK lysis buffer), depending on the pellet size. After incubation for 2 minutes at room temperature the ACK buffer was diluted 10-times with 1X PBS containing 2.5% FBS and pelleted again. Cell pellet was resuspended in 100 uL of 1X PBS + 2.5% FBS, mixed with 5 uL of Human TruStain FcX (Biolegend #422302), 3 uL of PE H2KD antibody 0.1 uL of calcein (1μg/μL, Calcein (Biolegend #425201)), and left for 15 minutes on ice. Stained samples were washed twice with 2 ml of 1X PBS + 2.5% FBS, and finally resuspended in the same buffer supplemented with DAPI dye. Using BD FACSAria (BD Biosciences) or Sony MA900 (Sony) flow cytometers, cells were sorted on DAPI-, Calcein+ (FITC+) to select for live cells. In addition, we sorted only H2KD- (human cells). FACS-sorted cells were subjected to scRNA-seq protocol using Chromium (10X genomics) instrument and Single Cell 3' Reagent Kit (v3). Each sample, containing approximately 3000-8000 cells was encapsulated and barcoded following the manual (CG000183 Rev B). The viability of samples varied between 58-98% (median 80%), as confirmed with 0.2% (w/v) Trypan Blue staining. The final sequencing libraries were double-size purified (0.6–0.8X) with SPRI beads and sequenced on Illumina Nova-Seq platform (R1 – 26 cycles, i7 – 8 cycles, R2 – 70 cycles or higher). On average, 3,330 cells per clinical sample (N = 62) were sequenced at a depth of ∼42.000 reads per cell (195 million reads per sample). The unique mapping was high, between 79-88%, and a median number of unique transcripts per cell was 4.393.