GSM7717047: WT_2; Mus musculus; RNA-Seq
To obtain tumour-filtrating immune cells, tumours were sliced into small pieces of 1-2 mm in petri dishes and then digested in digestion buffer (10 mM HEPES, 1.25 mg/ml collagenase D, 0.85 mg/ml collagenase Ⅴ, 30 U/ml DNase I, 1 mg/ml dispase Ⅱ, and 10% FBS diluted in HBSS) for 45 min on a rotor at 37℃. The dissociated cells were passed through 70 µm and 40 µm cell-strainers (BD) sequentially and centrifuged at 600×g for 5 min at 4°C, then prepared as single-cell suspension.  Tumour-inflitrating immune cells were enriched from the tumour tissues by magnetic separation using mouse CD45 MicroBeads (Miltenyi Biotec, cat. no. 130-052-301). Cellular suspensions were loaded onto a 10X Genomics GemCode Single-cell instrument to generate single-cell Gel Bead-In-EMulsion (GEMs).  Libraries were generated and sequencedfrom the cDNAs with Chromium Next GEM Single Cell 3'Reagent Kits v3.1.  Silane magnetic beads were used to remove leftover reagents and primers, and full-length, barcoded. cDNAs were amplified by PCR for library construction.  The libraries were constructed by adding specific primers during GEM incubation and library preparation steps, including End Repair, A-tailing, Adaptor Ligation, and PCR.