GSM8181844: MC38 shBST2, 10 tumors, SMCs; Mus musculus; RNA-Seq
Tumor tissue harvested from mice was placed in cold PBS prior to tissue digestion. Tumor tissue was mechanically dissociated into 2-4mm pieces and then digested in 2.5 mg/mL Liberase and 0.1 mg/mL Dnase (Roche) for 1 hour at 37C. Digested tissue was then passed through 70um filters and incubated with red blood cell lysis buffer for 10 minutes prior to neutralization with PBS. Cells were stained with a viability dye, spun down, and resuspended PBS +1% BSA. The first 3 libraries were composed of all live single cells from one dissociated tumor. Otherwise, live cells sorted for presence of fluorescent lineage tracer (either eGFP or tdTomato depending on mouse line). Fluorescent negative cells added back to final samples for sequencing as needed to ensure manufacturer-recommended concentration of 500 cells/uL minimum. Library was constructed according to the manufacturer's protocol (single cell 3' v3.1, 10X Genomics) by the Stanford Genome Sequencing Core. Briefly, cells were resuspended in the master mix and loaded together with the partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every GEM was reverse transcribed to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI), and the 10X barcode. The pooled and barcoded cDNA was then cleaned with Silane DynaBeads, amplified by PCR, and the appropriate sized fragments were selected with SPRIselect for library construction. During library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and a sample index were added.