GSM7567337: AireWT_GEX; Mus musculus; RNA-Seq
Thymi from 4 pooled 5 week old B6.Adig mice were dissociated by enzymatic digestion using Liberase TH  and DNase in RPMI at 37°C until complete digestion. The single cell suspension was then filtered through a 52 µm mesh filter and resolved on a Percoll gradient. The Percoll gradient was centrifuged at 2700rpm at 4°C with no break, for 30 minutes. Stromal cells where collected and washed followed by centrifugation.  Single cell suspensions were further enriched for thymic epithelial cells by staining for EpCAM-APC Cy7 followed by positive selection using anti-APC microbeads and the enriched fraction was stained for cell sorting. 60,000 viable (DAPI negative) CD45-EpCAM+ TECs were sorted into 500 ul PBS supplemented with 0.04% bovine serum albumin and then supplemented with 6,800 CD45-EpCAM+IA-IEloItgb4lo/neg cells. Cells were processed as per manufacturer's instructions for low cell input nuclei isolation. Briefly, cells were resuspended in 100 μl cold lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 0.1% NP-40 substitute, 0.01% digitonin, 1% BSA, 1 mM DTT, RNase inhibitor 1 U/µl) and divided into 2 tubes for 3 min lysis. Nuclei were then washed and resuspended in nuclei buffer to target 7,000 recovered nuclei. Nuclei transposition and mRNA barcoding were followed by library preparation per the manufacturer's (10x genomics) instructions which were sequenced with paired-end 100-bp reads on an Illumina NovaSeq6000. single cell 3' RNA-seq and single cell ATAC-seq