title: GSM5107421: tumor - HSF1-303-7; Mus musculus; RNA-Seq
library_construction_protocol: Tumors from HSF1S303/7A and WT mice were dissected, manually dissociated, and digested enzymatically with 0.04 U/mL Liberase TM (Sigma) and 0.15 mg/ml Dnase I (Roche) in Hank's Balanced Salt Solution (Life Technologies) for 30 min at 37 °C with agitation. Tumors were homogenized by briefly vortexing and filtered through a 70-μm nylon filter. Single-cell suspensions were washed with 1 × PBS-4% FBS before incubation 20 min on ice at 5 × 107 cells/ml with 500 ng/ml Fc block (2.4G2, BD Pharmingen) to prevent nonspecific antibody binding. Cells were then incubated 1 h on ice with PE-eFluor610-conjugated CD45 monoclonal antibody (30-F11, eBioscience). For scRNAseq libraries, DAPI- (live) CD45+ and CD45− cells were sorted using a SY3200 flow cytometer. Sorted cells were counted, and live CD45+ and CD45− cells were mixed 5:1. The libraries were prepared using the Chromium Single Cell 3′ Reagent Kits (v2, 10x Genomics): Single Cell 3′ Library & Gel Bead Kit v2 (PN-120237), Single Cell 3′ Chip Kit v2 (PN-120236) and i7 Multiplex Kit (PN-120262) and following the Single Cell 3′ Reagent Kits (v2) User Guide (manual part no. CG00052 Rev A). single-cell RNA-seq