title: GSM8333786: CBT_DEG_TdLN2; Mus musculus; RNA-Seq
library_construction_protocol: Prior to staining, cells were washed with FACS staining buffer (chilled PBS containing 1% FBS and 2 mM EDTA). Cells were resuspended in 50 mL of the antibody-containing staining buffer, plus eBioscience Fixable Viability Dye eFluor 780 or eFluor 506 to distinguish live and dead cells and with anti-CD16/CD32 (clone 93, BioLegend) to prevent non-specific antibody binding. Cell surface proteins were stained for 20 min on ice with fluorophore-conjugated antibodies at a 1:200 dilution. Cells were then washed twice and resuspended in eBioscience Fixation/Permeabilization buffer and incubated 30 minutes at room temperature. Cells were then washed twice and resuspended in staining buffer with intracellular antibodies. To obtain absolute counts of cells, Precision Count Beads (BioLegend) were added to samples according to manufacturer's instructions. Flow cytometry sample acquisition was performed on a LSR Fortessa cytometer (BD), and the collected data was analyzed using FlowJo v10.5.3 software (TreeStar). For cell sorting, the surface staining was performed as described above under sterile conditions, and cells were acquired and sorted into complete medium using a FACSAria III sorter (BD). For CD8+ T cell analysis, cells were pre-gated on FSA and SSC, Live, CD45+, CD45-IV-, CD3+ or TCRbeta+, single cells, CD4-, CD8+. Antibodies used: CD4 clone RM4-5, CD45 clone 30-F11, CD45.1 clone A20, CD45.2 clone 104, CD8a clone 53-6.7, TCRb clone H57-597, CD3 clone 17A2, Thy1.2 clone 53-2.1, CXCR3 clone CXCR3-173, CX3CR1 clone SAO11F11, KLRG1 clone 2F1/KLRG1. SIY-tetramer was obtained from the NIH tetramer core, conjugated to PE, and added at 1:500-1:1,000 to the extracellular staining antibody mix. For scRNA-seq experiments with cell hashing, cells were also stained with Totalseq A anti-mouse hashing antibodies (Biolegend) before FACS. Sorted cells were then processed for scRNA-seq using the Seq-Well platform with second strand chemistry, as previously described (63, 64). Whole transcriptome libraries were barcoded and amplified using the Nextera XT kit (Illumina) and were sequenced on a Novaseq 6000 (Illumina). Hashtag oligo libraries were amplified as described previously and were sequenced on a Nextseq 550(38).