GSM8602775: Mouse melanoma Cd45 cells, 20M; Mus musculus; RNA-Seq
4-, 12-, and 20-months old mice (7 mice for each age) were sacrificed 18 days after tumor cell inoculation. Tissues were excised, minced, and digested with 1 mg/ml collagenase D (Roche) and 100 µg/ml DNase I (Sigma) at 37°C for 45 minutes. Digests were then passed through a 70-μm cell strainer to generate a single-cell suspension. Single cell suspensions were washed with 1XPBS-4%FBS before incubation for 20 min on ice at 5•107 cells/ml with 500 ng/ml Fc block (2.4G2, BD Pharmingen). Cells were then incubated for 1 hour on ice with AF700 conjugated CD45.2 monoclonal antibody (104, Biolegend). For scRNA-seq libraries, DAPI negative (live) CD45+ cells were sorted using a flow cytometer, and sorted cells were resuspended in RPMI for counting. Libraries were prepared using the Fluent Biosciences PIP-seq v4.0 Plus T20 3' Single Cell RNA kit according to the User Guide. Libraries were sequenced on an Element Biosciences AVITI sequencer with 60x100bp sequencing to a read depth of 20k – 27k reads per cell.