GSM7823242
Lung tissue was perfused with 5 mL cold sterile heparinized PBS by injection into the right ventricle. The lungs were then minced, placed in a Miltenyi C-tube containing digestion buffer (432 U/mL collagenase IV, 64 U/mL DNase), and then dissociated for 30 min on the Miltenyi Gentle MACS  OctoDissociator.  Samples were then filtered and prepared for Dead Cell removal (Miltenyi). Following dead cell removal, mouse immune cells were isolated using the CD45+ cell microbead isolation kit (Miltenyi) following manufacturer's instructions. After counting, single cell suspensions were prepared in cold buffer (1X PBS with 2% BSA). The library was generated using the 10X genomics 3'-seq kit v3.1 following manufacturer's protocol. Single cells were captured using the Chromium Next GEM single cell 3' Kit v3.1, 16 rxns PN-1000268, Chromium Next GEM Chip G Single Cell Kit, 48 rxns PN-1000120 and Dual Index Kit TT Set A, 96 rxns PN-1000215 (all from 10x Genomics) and run using Protocol CG000315 Rev C.