GSM4656014: Gene expression replicate 2 run 2; Mus musculus; OTHER
Freshly isolated thymocytes from three-week-old Rag2-Gfp Foxp3-Thy1a B6 mice were stained with the fluorochrome-labelled and nucleotide-labelled antibodies listed in Table S2C. GFP+CD4+CD8-S1P1+Thy1.1+ Tregs were FACS sorted. Single-cell libraries were generated using the Chromium Controller Single-Cell Instrument and Chromium Single Cell 3' Library & Gel Bead Kit v2, A Chip Kit and i7 Multiplex Kit according to the manufacturer's protocol. 15000 cells per sample (n=3) were injected into each channel and samples were run according to manufacturer's protocol with a targeted cell recovery estimate of 24000 cells in total. Cellular suspensions were loaded on a GemCode Single-Cell Instrument (10x Genomics) to generate single-cell GEMs. Each droplet contained therefore antibodies and cDNA molecules that are indexed with the same unique barcode and these were converted into libraries that were amplified independently and mixed at 1:4 proportions for sequencing a total of three runs. Sequencing libraries were cleaned up with SPRIselect beads and then loaded on a HiSeq 3000 instrument (Illumina) for sequencing with aforementioned proportions.