GSM6214550: t_cell_res; Mus musculus; RNA-Seq
Single-cell suspensions of tumor cells were prepared by passing the tumor through a syringe without a needle followed by a 1 h digestwith shaking at 37◦C in 100μg/mL DNase I (Roche, Basel, Switzerland; catalog no. 11284932001),300 U/mL collagenase/100 U/mL hyaluronidase (Stemcell Technologies, Vancouver, Canada; catalogno. 07912), 0.6 U/mL Dispase II (Roche, Basel, Switzerland; catalog no. 4942078001) in DMEM/D12with 10% FBS (Gibco, Waltham, MA, USA). Digests were filtered through a 100μm cell strainer prior to library construction. For T cell sequencing, Immune and cancer cell depletions were performed for T cell specific reactions using Pan T Cell Isolation Kit II, mouse (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat# 130-095-130) in combination with CD90.1 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat# 130-121-273) per manufacturers protocols prior to were cell depletion using LS columns (Miltenyi Biotec, Bergisch Gladbach, Germany; Cat# 130-042-401). Cells were resuspended in PBS with 0.04% non-acetylated BSA prior to the single-cell sequencing step using Chromium Single-cell 3ʹ GEM, Library & Gel Bead Kit v3 (10× Genomics) on a 10x Genomics Chromium Controller following manufacturers protocol. Single-cell library preparation was performed using Chromium Single Cell 3′ GEM, Library & Gel Bead Kit v3 (10x Genomics, Pleasanton, CA, USA; Catalog no. 1000075) following the manufacturer's protocol.