title: GSM7680409: scRNA-seq GEX library; Mus musculus; RNA-Seq
library_construction_protocol: Detailed description of tissue digestion and sorting available in linked publication Target cells from spleen (12,500 CD3+CD4+CD25+ Treg cells, TotalSeqC1), mesenteric (10,000 CD3+CD4+CD25+ Treg cells, TotalSeqC2), inguinal lymph nodes (7,500 CD3+CD4+CD25+ Treg cells, TotalSeqC3) and skin (10,000 CD3+ T cells, TotalSeqC4) have all been sorted in a single 1.5 mL Eppendorf tube containing 350 μL MACS buffer, and the sample collection tube was cooled to 4°C. It is important to process the sample quickly after sorting to decrease the number of dying cells, which would again increase the count for mitochondrial DNA in the sequencing data. Therefore, shortly after sorting, cells are pelleted by centrifugation (5 min, 300 xg, 4°C). Supernatant is removed and the sample is supplemented with master mix and beads to a final volume of 70 μL, loaded on a 10X Chromium Next GEM Chip K (10X Genomics #1000287) and processed on the 10X Chromium Controller (10X Genomics #120212), followed by cDNA amplification using the Chromium Next GEM Single Cell 5' Reagent Kit v2 (10X Genomics #1000263) and 5' Feature Barcode Kit (10X Genomics #1000256). V(D)J amplification was done from cDNA by using the Chromium Single Cell Mouse TCR Amplification Kit (10X Genomics #1000254) and GEX, CSP and VDJ library preparation according to the Library Construction protocol (10X Genomics #1000190).