GSM7230111
To isolate T cells from collagen gels, collagen matrices were mechanically chopped into small pieces and digested in 100U/ml collagenase type IV (ThermoFisher Scientific #17104019) at 37ºC for 1 hour in digestion media (RPMI 1640 + 10% FBS), with intermittent mechanical disruption through 1ml pipette tips. Collagenase was then quenched with MACS buffer (PBS, 0.5% BSA, 2mM EDTA), after which cells were then passed through 30µM strainers (Miltenyi #130-098-458) and washed twice in PBS. Live cells were enriched using a dead cell removal kit (Miltenyi #130-090-101) according to the manufacturer's protocol and resuspended in PBS+2% BSA Library construction was performed by the Bauer Core Facility at Harvard University according to the manufacturer's protocol (10x Chromium Single Cell 3' v3 platform)