title: GSM9435484: HY19636 tumor, D14; Mus musculus; RNA-Seq
library_construction_protocol: Mice were euthanized and tumor mass was carefully excised from the subcutaneous region without collecting the adjacent skin and surrounding adipose tissue. On ice, samples were finely minced with scissors and then placed in a 2 ml tube containing 1.5 ml of digestion medium (2 mg/ml collagenase XI (Millipore Sigma, C7657), 0.5 mg/ml hyaluronidase (Worthington, LS005477), 0.1 mg/ml DNase (Roche, 10104159001) in DMEM (Gibco, 11995065) supplemented with 10% FBS (Foundation C, Gemini Bio, 900-308), 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 50 µM beta-mercaptoethanol (Gibco, 21985-023). The tube was placed horizontally in a bacterial shaker for 45 min at 37ºC and 225 rpm. The tumor samples were then filtered through a 40 µm filter. Samples were then washed with 10 ml cold PBS. Prior to sorting of cells on on a BD FACSAria II cell sorter (BD Biosciences), cells were stained with Zombie-NIR (BioLegend, 1:200) and  Calcein blue AM (Thermo Fisher Scientific, C1429, 10 µM working concentration). Samples were additionally stained with anti-mouse CD45-BUV395 (clone 30-F11, BD Biosciences, 1:500), anti-Thy1.2/CD90.2-BV786 (clone 53-2.1, BD Biosciences, 1:500, for Thy1 detection), and anti-E-Cadherin/CD324-PE-Cy7 (clone DECMA-1, BioLegend, 1:200). Four live cell (Zombie-NIR-negative, Calcein blue AM-positive) populations were sorted: (1) CD45+ (immune cells), (2) CD45- EGFP+ Thy1+ (fibroblasts), (3) CD45- EGFP- E-Cadherin- (miscellaneous), (4) CD45- EGFP- E-Cadherin+ (tumor cells). The four populations from each sample were then pooled in a 15 ml Falcon tube and pelleted for 7 minutes at 500 g. Cells were processed using the Chromium Fixed RNA Profiling (Gene Expression Flex) pipeline. In brief, each sample was first fixed using the Single Cell Fixed RNA Sample Preparation Kit (10x Genomics, PN-1000414) following the manufacturer's instructions. Samples were fixed for 18 h at 4 °C and, after addition of quenching buffer and storage buffer, stored at -80 °C until further processing. After thawing of samples, samples were processed using the Chromium Fixed RNA Kit, Mouse Transcriptome (10x Genomics, PN-1000496) following the manufacturer's instructions to generate a fixed RNA gene expression library. For multiplexing, each sample (4 replicates * 4 AAV groups = 16 total) was hybridized with a separate Probe Barcode library including added custom probes (Egfp, 8casd09, GGTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATC; and Egfp, 8casd10, AGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTC). Approximately 2.5e5 total cells were encapsulated and sequenced on two lanes of a NovaSeq X flow cell (1.25 B reads/lane) to reach about 10.000 reads/cell.