GSM8396154: CD45+, tumor, PKCd KO BM, Replicate 2; Mus musculus; RNA-Seq
After 26 days of growth tumors were excised from WT or PKCd KO bone marrow engrafted animals, mechanically dissociated, and incubated with a mixture of 10 mg/ml collagenase type IV (Sigma, C5138), 1 mg/ml hydrogenase type V (Sigma, H6254), and 2,000 units/ml DNAse type IV (Sigma, D5025-150ku) for 45 minutes at 37C before being washed with PBS through a 70uM filter (Corning, 352350). Tumors were then passed through a Ficoll-Paque gradient (Cytivia Life Sciences, 17144003), and the bilayer was collected. Following the ficoll gradient, the mononuclear cells obtained from two tumors were combined into a single sample forming a single replicate for that group. Samples were then stained with Live-Dead (APC-Cy7) and CD45.2 (FITC) and Live CD45.2+ cells were sorted via fluorescence activated cell sorting (FACS) before being washed 3x in PBS. 2 WT and 2 PKCd KO samples were processed simultaneously. This entire process was repeated on a second, independent collection day, for a total n of 4 replicate samples from 8 total tumors per genotype group. Single cell partitioning and library creation was performed by the University of Chicago Genomics core using a chromium controller from 10x genomics following the “Chromium Single Cell 3ʹ Reagent Kits v3” user guide and associated reagents. 10,000 cells per sample were targeted for encapsulation. Libraries were sequenced in PE100 format of an SP-100 flowcell of an Illumina Novaseq 6000 instrument.