title: GSM6883043: tumor, day 18,  replicate 2, scRNAseq, animal ID 629-5; Mus musculus; RNA-Seq
library_construction_protocol: Tumor samples were collected on days 11, 14, 18, and 24 post-tumor initiation (Figure 1A). Animals were sacrificed by lethal anesthesia with an intraperitoneal injection of pen- tobarbital (150 mg/kg). Tumors were localized in the fourth mammary fat pad by palpation and visual inspection.  Using a scalpel, 10-250 mg of tumor tissue was cut for single-cell RNA sequencing. The tissue was further cut into 1-2 mm-sided cubes. The cubes were positioned on a 40 μm filter and washed with 15 mL Hanks' Balanced Salt Solution (HBSS). Tumor fragments were collected into C-tubes (Miltenyi, Switzerland) using a pipette tip, and 4.7 mL of DMEM Glutamax w/o FBS were poured over the reversed filter to detach any small remaining fragments into the C-tube. Enzymes H (200 μL), A (25 μL), and R (100 μL) from the GentleMACS tumor dissociation kit (Miltenyi, Switzerland) were added to the C-tube. C- tubes were processed using the human tissue dissociation kit program 1 for soft tissues on a GentleMACS OctoDissociator instrument (Miltenyi, Switzerland) for 65 minutes. The dissociated tumor tissue was filtered through a 40 μm filter into a 50 mL falcon by pre-washing with 10 mL of HBSS, pouring the dissociated tumor tissue on the filter, using a syringe plunger on the filter to gently squeeze tissue through the filter, and washing the filter with 5 mL HBSS to yield 20 mL volume of dissociated tissue. Dissociated cells were pelleted at 300xG for 10 min at 4 degrees Celsius. The supernatant was discarded, and pellets were re-suspended in 10 mL 1x red blood cell lysis buffer and incubated at room temperature for 10 min. Cells were pelleted again at 400xG for 10min at 4 degrees Celsius, re-suspended in 5 mL of filtered DMEM w/FBS, and counted twice on a Countess automatic cell counter (ThermoFisher Scientific). Viability was quantified by Tryptan Blue staining. Cell counts and viability were estimated by averaging the two measurements. Cells were checked for the absence of significant doublets or aggregates and loaded into a Chromium Single Cell Controller (10x Genomics, Pleasanton, CA) in a chip together with beads, master mix reagents (containing RT enzyme and poly-dt RT primers), and oil to generate single-cell-containing droplets. Single-cell Gene Expression libraries were then prepared using Chromium Single Cell 3' Library & Gel Bead Kit v3.0 (PN-1000075) following the manufacturer's instruction (protocol CG000183 Rev C). Quality control of the libraries was performed with a TapeStation 4200 (Agilent) and QuBit dsDNA high-sensitivity assay (Thermo Fisher Scientific) following manufacturer instructions. With this procedure, the cDNAs from distinct droplets harbor a distinct and unique 10x cell barcode.