scRNA-seq of Mus musculus CD4+Tregs
T cells were sorted from LI 7 to 8 weeks after adoptive T cell transfer as CD45+TCR+CD45.2+Foxp3/GFP+ or CD45+TCR+CD45.1+Foxp3/hCD2 Tregs, or CD45+TCR+CD45.1+ Teff with FACSAria II (Becton Dickenson). Sorted cells were encapsulated into droplets with the ChromiumTM Controller (10x Genomics) and processed following manufacturers specifications. Transcripts captured in all the cells encapsulated with a bead were uniquely barcoded using a combination of a 16 bp 10x Barcode and a 10 bp unique molecular identifier (UMI). cDNA libraries were generated using the Chromium Next GEM Single Cell 5 Reagent Kit v1 or v2 (10x Genomics) following the detailed protocol provided by the manufacturer. Libraries were quantified by Quantus (Promega) and quality was checked using TapeStatoin 4200 with High Sensitivity DNA kit (Agilent). Libraries were sequenced with NextSeq 500 platform (NextSeq 500/550 Mid Output Kit v2.5 (150 cycles), Illumina)  with paired-end mode or NovaSeq 6000 platform (SP Reagent Kit v1.5, Illumina) in 50 bp paired-end mode. The hashtag library was demultiplexed using CellRanger software (version 7.1.0). Raw data was demultiplexed with cellranger mkfastq (10x Genomics v7.1.0) to generate fastq files.