title: GSM7809110: Thymic hematopoietic non-T cells, 18h Dex1+4d dex2, GEX; Mus musculus; OTHER
library_name: GSM7809110
library_construction_protocol: Mice were sacrificed and perfused, thymi were surgically removed, trimmed of fat and connective tissues, and placed in PBS on ice. Thymi were disintegrated by enzymatic digestion for 30 min in a 37°C water bath, using 0.016 mg/ml Liberase TM (Roche, 5401127001) and 10 ng/ml DNase I (Sigma-Aldrich, DN25) in RPMI, then filtered through a 50-μm mesh filter. Cells were washed with 5–10 ml MACS buffer. 5 million cells per sample were first stained with 5 μl anti-mouse CD16/CD32 monoclonal antibody (Biolegend, 101302) in 50 μl labeling buffer (PBS + 1% BSA) for 10 min at 4 °C, then an antibody mix containing both TotalSeq-B and flow cytometry antibodies in labeling buffer was added to complete to ~100 μl total volume, and cells were incubated for 30 min at 4 °C. TotalSeq-B antibody mix was prepared in advance per sample in labeling buffer (PBS + 1% bovine serum albumin, BSA) at a total volume of 50 μl and centrifuged at 14,000 x g for 10 min at room temperature, prior to its combination with the flow cytometry antibody mix and incubation with the cells. Cells were then washed 3 times with 5 ml labeling buffer. 10 minutes before each sample was sorted, it was incubated with a viability dye for 5 min at room temperature, then washed and resuspended in PBS + 2% BSA. Cells were sorted into PBS supplemented with 50% FBS and then washed and resuspended in PBS + 0.04% BSA, counted on a hemocytometer, and volume was adjusted to a concentration of 1,000,000 cells/ml. Each two non-replicate samples were combined evenly into one pool. scRNA-Seq and CITE-Seq libraries were prepared using the Chromium 10x Genomics platform and Next GEM Single Cell 3ʹ Reagent Kits v3.1 (Dual Index). Loading and library preparation were done according to the manufacturer's instructions (10X genomics).