title: GSM8728842: Control treated tumours, individually hashtagged and pooled [SZ26_01_10X_S1]; Mus musculus; RNA-Seq
library_name: GSM8728842
library_construction_protocol: Tumors were collected into complete RPMI on ice, cut into small pieces and digested with Collagenase IV (200 U/ml, Worthington Biochemical) and DNase I (0.2 mg/ml, Roche) for 30 min at 37°C, vortexing once. The tumors were filtered through a 70µm cell strainer and pelleted. Cell pellets were resuspended in FACS buffer. Fc receptors were blocked with anti-CD16/CD32 (clone 93, eBioscience) 5 min before staining. Tumors were stained with Aqua LIVE/DEAD-405 nm (Invitrogen) and CD45-BV605 (Clone 30-F11) antibodies. Each tumor (n=5 per group) was stained with a TotalSeqTM HTO barcode (BioLegend) prior to sorting.  FACS buffer used in these experiments was EDTA and sodium azide free. Live CD45+ cells were sorted on a BD FACSAria III with a purity > 98%. Cells were counted using a hemocytometer after Trypan Blue exclusion. 17,100 (sample 1) or 3,500 (sample 2) live cells were loaded into a channel of a Chromium Next Gel Bead-in Emulsion (GEM) Chip G (10X Genomics, PN-1000120) and GEMs were generated on the Chromium X (10X Genomics). Indexed sequencing libraries were prepared using the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (10X Genomics, PN-1000268) according to BioLegend Totalseq-A protocol and 10x Genomics user guide, with 11 cycles of cDNA amplification and 15 cycles of sample index PCR.
identifiers.namespace: GEO
identifiers.id: GSM8728842_r1