title: GSM8000270: TCR; Mus musculus; OTHER
library_construction_protocol: SI-IELs or SP cells were collected and pooled from young (6-week-old) or old C57BL/6 mice (80-week-old), the cells from 3 young mice and 3 old mice were mixed and pooled as young SP, young IEL, old SP and old IEL. CD45+ cells were enriched from each group using CD45 microbeads (Miltenyi Biotec), followed by cell hashing with TotalSeq-C anti-mouse hashtag antibodies (hashtag1-young IEL, hashtag2-old IEL, hashtag3-young SP, hashtag4-old SP). After the Fc blocking, cells were stained with anti-CD3e-FITC mAbs and anti-CD4-APC mAbs, and the CD3+ cells and CD3+CD4+ cells were sorted from each group by FACSMelody. Dead cells removal was performed with 7-AAD. To enrich CD4+ cells, which are rare population in young IEL, CD3+ cells and CD3+CD4+ cells were mixed as 1:1 in young and old IEL. Cell suspension (6,000 cells each) was loaded in a 10x Chromium microfluidics system based on the manufacturer's guidelines. One set of libraries were obtained from the 10x loaded samples: a 5' gene expression messenger RNA library and a single-cell TCR library, using primers for amplification as per the manufacturer's instructions. Library were pooled together and run separate lanes of 150 base-paired, paired-end, flow cell using the Illumina Novaseq6000.