GSM8151507: BC17122, scRNAseq; Homo sapiens; RNA-Seq
Breast cancer tissues were placed in RPMI 1640 medium (11875093, Thermo Fisher Scientific, Waltham, MA, USA) and promptly transported to the laboratory within two hours post-resection. After washing with Dulbecco's Phosphate Buffered Saline (DPBS, L0615, Biowest, Nuaillé, France) containing 1× ZellShield anticontaminant agent (Minerva Biolabs, Berlin, Germany), the tumour tissues were minced into 1-mm-diameter pieces. The minced breast cancer tissue was incubated for 1 h in digestion buffer (DMEM-F12 (11330032, Thermo Fisher Scientific), 2% FBS (Corning, VA, USA), 1% penicillin/streptomycin (15140-122, Thermo Fisher Scientific), 10 μg/mL insulin (51500056, Thermo Fisher Scientific), and 10 ng/mL epidermal growth factor (PHG0311, Thermo Fisher Scientific) supplemented with 1× collagenase/ hyaluronidase (CA094-002, Gendepot, Barker, TX, USA). The digested samples were centrifuged at 80 × g for 30 s, and the resultant supernatant was filtered through a 70-μm nylon mesh strainer (352350, Corning Life Sciences, Tewksbury, MA, USA). The pellets from the digested samples were resuspended in 0.25% trypsin/EDTA (25200072, Thermo Fisher Scientific) and incubated for 3–5 min to release single cells. Once a single cell suspension was obtained, cold Hank's balanced salt solution (L0612, Biowest) containing 2% FBS was added, and cells were recovered through centrifugation at 300 × g for 5 min. Subsequently, the cells from the supernatants and the single cells from the pellet were combined, counted, and cryopreserved. CD45+ cells were isolated either through magnetic bead sorting or fluorescence-activated cell sorting (FACS). In the magnetic separation method, dead cells were initially removed using a dead cell removal kit (130-090-101, Miltenyi Biotec, Auburn, CA, USA), followed by cell labelling with CD45-microbeads (130-045-801, Miltenyi Biotec) and enriched using a magnetic-activated cell sorting (MACS) column (130-042-901, Miltenyi Biotec) and MACS separator (130-091-051, Miltenyi Biotec). For FACS sorting, single cells were stained with an anti-human CD45 antibody (304016, Biolegend, San Diego, CA, USA) and resuspended in 4',6-diamidino-2-phenylindole solution (D1306, Thermo Fisher Scientific). CD45+ live cells were sorted using FACSAria (BD bioscience, San Jose, CA, USA). The isolated cells were resuspended in CS10 freezing media (100-1061, Stemcell Technologies, Bothell, WA, USA) and stored in an NL2 tank until the next use or were immediately sequenced scRNA-seq library preparation was conducted using the Chromium Single Cell 3′ Kit v2 and the Chromium Next GEM Single Cell 3′ Kit v3 platform (10x Genomics, Pleasanton, CA, USA)