GSM6214648: Myeloid specific c-Rel KO; Mus musculus; RNA-Seq
To obtain tumor-infiltrating cells, tumors were digested in RPMI 1640 (Gibco) supplemented with 0.5 mg/ml collagenase type IV (Gibco) and 0.1 mg/ml DNase I (Sigma-Aldrich) for 20 min at 37°C. Tumors were then treated with Percoll(GE Healthcare Lifesciences, 17089101) and immune cells were isolated for flow cytometry and sorting. FACS-isolated CD45+ cells from the 5 pooled tumors from WT (LysM-cre x Rel+/+) or myeloid Rel knockout mice (LysM-cre x RelloxP/loxP) were washed once in PBS (calcium- and magnesium-free) containing 0.04% BSA and resuspended to a concentration of 700-1200 cells/ul. The DNA libraries were prepared using the Chromium Single Cell 3′ Reagents Kits and 10x Genomics Chromium platform for droplet-enabled scRNAseq according to the manufacturer's instructions. Each library was sequenced on the Illumina HiSeq 4000 platform to achieve an average of 10000 reads per cell.