GSM8328319: spleen-WT-CD45_CD11b; Mus musculus; RNA-Seq
After gating live singlets, CD45+ CD11b+ myeloid cells were isolated from dissociated spleens, and CD45+ immune cells were isolated from dissociated tumors using a BD FACS Aria II using the purity setting. Equal numbers of cells were sorted per mouse from 3-4 mice into one pooled sample per genotype for spleens and for tumors. After sorting, samples were centrifuged at 500g for 5 min. Supernatant was removed to leave 25ul of solution to not disturb pellets. Remaining volume was measured and raised to 50ul total by adding a 1:1 mixture of cold FACS buffer and nuclease-free water. 16,000 cells were loaded per 10x lane (Chromium Single Cell 3' Reagent Kit v3.1). Single cell RNA-seq library and transcriptome library construction were prepared according to the 10x Chromium Single Cell 3' v3.1 user guide (Rev A). Indexed and pooled single cell transcriptome libraries for each mouse were sequenced on the NovaSeq 6000 System with an S1 100-cycle kit.