title: GSM8122271: Vehicle, 24 hours post-treatment, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Tumors were initially minced with scissors and resuspended in a 0.8% Collagenase A solution (in serum-free DMEM) followed by dissociation for 40 minutes, 37°C with gentle agitation using the gentleMACS dissociator (Mitenyi Biotech, San Diego, CA). Upon digestion, samples were neutralized with the addition of 2 volumes of DMEM (10%FCS) and strained through 70μm and 22μm filters prior to centrifugation at 4°C, 400rpm for 2 minutes. Red blood cells were removed from the samples by the addition of a red cell lysis buffer (150 mmol/L NH4Cl, 1 mmol/L KHCO3, 100 mmol/L EDTA, pH 8) for 2 minutes, and then clarified by centrifugation. Samples were resuspended in DMEM 2% FCS and evaluated for viability by trypan blue staining and hemocytometer count. Dissociated viable cells (3x106) were pooled from three independent tumors per treatment, labelled with Sytox Green viability dye (Thermo Fisher, Waltham, MA). Viable cells (20,000) were recovered by flow cytometry (BD FACS Aria; BD Biosciences, Franklin Lakes, NJ) and loaded onto a Chromium Single Cell Chip (10x Genomics, Pleasanton, CA). The cDNA preparation was prepared according to the Single Cell 3′ V 3.0 Protocol.The barcoded cDNA was purified using solid‐phase reversible immobilization (SPRI) beads and amplified in a PCR step. The Read 1 (R1) primer was added during the Gel Beads-in-emulsion (GEM) generation. The P5, P7 and Read 2 (R2) primers were added via end repair, A‐tailing, adaptor ligation, and PCR. The sequencing was completed on an Illumina Nextseq500 platform (Illumina, San Diego, CA) using recommended sequencing parameters.