GSM9402202: Lung resident CD8+ T cell hastag oligos (HTO) for multiplexing; Mus musculus; OTHER
Prior to sacrifice animals were anesthetized and administerd 3ug of fluorescently labeled anti-mouse CD45 BV421 (clone 30-F11) in 150 uL PBS. After 3 minutes of antibody circulation mice were euthanized with isofluorane. After cardiac perfusion with PBS, lungs were removed from the chest and chopped for approximately 1 minute with a razor blade to dissociate the tissue. Chopped lungs were then incubated in a digestion cocktail of Collagenase IV (125 units/mL), DNase (50U/mL) and 1x Penicillin-streptomycin in HBSS (with Ca2+Mg2+) and incuated for 1 hour at 37C with continuous shaking. The mixture was then washed with FACS buffer (2% FBS and 1% Penicillin-Streptomycin in DMEM) and passed through a 70 μm cell strainer. Cells were pelleted at 550xg for 5 minutes and then resuspended in red blood cell (RBC) lysis buffer for 2 minutes. After RBC lysis cells were passed through a 40 μm cell strainer. Cell suspensions were blocked with mouse FcR blocking reagent (Miltenyl Biotec 130-092-575) for 15 minutes and then incubated with the appropriate fluorescently-conjugated antibodies and Biolegend barcode antibodies in sorting buffer for 30 min at 4C. After washing with FACS buffer cells were sorted. Doublets and dead cells were excluded based on forward and side scatter and Sytox Red, respectively. The following antibodies were used for staining: anti-CD45 UVC (BD 564279),  anti-CD3 FITC, anti-CD4 BV711, anti-CD8 BV786. Endogenous tdTomato was used to identify Gzmk+ cells within the CD8+ population. FACS was performed on a BD FACS Aria using FACSDiva Software. cDNA libraries were prepared for sequencing using standard protocol for the 10x Single Cell instrument with 5' v3 GEM-X technology with feature barcode and TCR.