title: GSM8542367: Spleen, immune cells, GEX, Inlet3; Mus musculus; RNA-Seq
library_construction_protocol: Spleens were harvested from male, 8 week-old CD45.1 recipients of CD45.2 Tet2/3 DKO T cells after 2 days (3 replicates) and 10 days (3 replicates) post transfer. Splenocytes were dissociated and 2 million cells were stained with fixable viability dye (dilution 1:1000), anti-CD45.2 PE (clone: 104, RRID: AB_313445, Biolegend) and anti CD45.1-BV421 (clone: A20, RRID: AB_10896421, Biolegend). To allow multiplexing of cells, splenocytes from each condition were stained with TotalSeq antibodies (Biolegend) and were incubated for 30 minutes on ice Cells were washed 3 times to allow removal of excess antibodies. After sorting, CD45.1+ cells were provided to the UNC Advanced Analytical core for library preparation. Cells were counted and viability was assessed using a Luna-FX7 automated cell counter (Logos biosystems).  Briefly, cell viability was assessed and 5.000 per biological replicate and per condition were pooled yielding a total of 30,000 cells (15,000 for day 2 and 15.000 for day 10 post transfer). 10.000 cells were loaded in 10x Genomics Chromium Single Cell 3' inlets (three inlets in total were used). Single-cell gel beads in emulsion (GEM) were generated.