title: GSM8059844: SW480-CBC subcutaneous tumor, replicate 2, scRNAseq; Homo sapiens; Mus musculus; RNA-Seq
library_construction_protocol: Xenograft tumors were processed and sorted for live cells to generate single cell libraries for mRNA sequencing. In brief, xenograft tumors excised from mice were weighed before immediate mechanical and enzymatic dissociation in DMEM-collagenase media for 1 hour in a 37˚C shaker at 150 rpm (Hyclone DMEM, 2 mg/mL Collagenase type IV (Sigma), 5% FBS (Atlas Biologicals), 1% Antibiotic-Antimycotic (Gibco); for orthotopic tumors, 20 ug/mL Hyaluronidase (Sigma) was also added). After dissociation, samples were centrifuged at 500xg for 5 min at 4˚C, washed with Hank's balanced salt solution (HBSS) and centrifuged again. Pellets were resuspended in TrypLE Express Enzyme (Gibco) and incubated at 37˚C for 6 min, pipetting with a p1000 pipet every 2 min to break cell clumps and adding DNase (100µg/mL = 0.3units/µL) as needed. DMEM containing FBS was used to quench samples before filtering with a 40µm strainer followed by centrifugation at 500xg for 5 min at 4˚C. Samples were washed with FACS buffer (1% BSA in PBS), centrifuged again, and cell pellets resuspended in FACS buffer for 2 million cells/100µL before adding SYTOX blue viability dye (1:10,000). Live cells (SYTOX blue-negative) were sorted using a BD FACSAria Fusion cytometer and then immediately washed with 0.04% BSA in PBS twice before a final resuspension to approximately 1,000 cells/µL for droplet-based library generation and sequencing. Each xenograft sample was generated as an individual scRNA-seq library using the Chromium Single-Cell 3'RNA-sequencing platform with Reagent kit v3 (10X Genomics) following manufacturer's protocols. Sequencing of each scRNA-seq sample was performed to achieve a read-depth of approximately 50,000 reads per cell.