scRNA-seq of tumor infiltrating CD11c + cells in ItgaxΔPtpn6 model
Subcutaneous MC38 tumors were generated in female mice and harvested on day 12 after tumor inoculation. Tumors from five mice per genotype were pooled, mechanically dissociated, and enzymatically digested with 0.5 mg/mL collagenase IV and 0.2 mg/mL DNase I for 1 hour at 37°C. Single-cell suspensions were filtered, red blood cells were lysed, and CD11c+ tumor-infiltrating cells were enriched by magnetic positive selection and subsequently FACS-sorted before single-cell RNA-seq.  No separate bulk RNA extraction was performed. Purified viable CD11c+ cells were loaded directly into the BD Rhapsody Single Cell Analysis System, where individual cells were captured in nanowells, lysed, and released mRNA was captured on barcoded beads for downstream cDNA synthesis.  For each sample, 40,000 cells were loaded into a BD Rhapsody Single Cell Analysis System cartridge. After cell capture, lysis, and mRNA capture on barcoded beads, cDNA synthesis was performed according to the manufacturer's instructions. Independent indexed whole-transcriptome libraries were then prepared for each sample. Library size was assessed using an Agilent 2100 Bioanalyzer and concentration was measured with a Qubit fluorometer. 