GSM5857048: mouse_expt_arms_1_2_3_4_5_gex; Mus musculus; RNA-Seq
For the mouse studies, CD44+CD4+ T cells from the indicated arms of mice were sorted using a BD FACSAria sorter. Cells from different treatment arms were labeled with hashtag antibodies, as well as CITE-Seq antibodies to CD25, OX40, TIGIT, PD1, Tim3, LAG3 (Biolegend). Sorted CD44+CD4+ T cells were resuspended at 106 ml in 50% FBS in 1×PBS and captured using 10x Genomics Single Cell 5' Solution. Single cell TCR sequencing was performed using the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 (10x Genomics) according to the manufacturer's instructions. For the human studies, cells were sorted using a BD FACSAria sorter. Ten-thousand cells from each of the three sorted fractions were pooled together and resuspended at 10^6 ml in 50% FBS in 1×PBS before capture using 10x Genomics Single Cell 5' Solution. Single cell TCR sequencing was performed using the Chromium Next GEM Single Cell V(D)J Reagent Kits v1.1 (10x Genomics). For 10X Genomics scRNA-seq, we generated three libraries that measure: (1) mRNA transcript expression (GEX), (2) sample/tissue-specific hashtag oligos (HTO) and protein marker levels using antibody derived tags (ADT), and (3) T cell receptor specific mRNA reperitorires. Cells were harvested and stained as described above. The sample was split into three libraries (GEX, HTO/ADT, and TCR). Reverse transcription PCR and library preparation were carried out under the Chromium Single Cell 5' v1.1 protocol (10X Genomics) per manufacturer's recommendations. The libraries were sequenced on the NovaSeq 6000 with 2x150 bp paired-end reads (Illumina).