title: GSM4085092: Week 6 anti-RANKL; Mus musculus; RNA-Seq
library_construction_protocol: Mouse thymi were isolated, cleaned of fat and transferred to DMEM (UCSF Cell Culture Facility) containing 2% FBS (Atlanta Biologics) on ice. For each scRNA-seq sample, three thymi were pooled. Thymi were minced with a razor blade and tissue pieces were moved with a glass Pasteur pipette to 15 ml tubes and vortexed briefly in 10 ml of media. Fragments were allowed to settle before removing the media and replacing it with 4 ml of digestion media containing 2% FBS, 100 ug/ml DNase I (Roche), and 100 ug/ml Liberase TM (Sigma-Aldrich) in DMEM. Tubes were moved to a 37°C water bath and fragments were triturated through a glass Pasteur pipette at 0 min and 6 min to mechanically aid digestion. At 12 min tubes were spun briefly to pellet undigested fragments and the supernatant was moved to 20 ml of 0.5% BSA (Sigma-Aldrich), 2 mM EDTA (TekNova), in PBS (MACS buffer) on ice to stop the enzymatic digestion. This was repeated twice for a total of three 12 min digestion cycles, or until there were no remaining tissue fragments. The single cell suspension was then pelleted and washed once in MACS Buffer. Density-gradient centrifugation using a three-layer Percoll gradient (GE Healthcare) with specific gravities of 1.115, 1.065, and 1.0 was used to enrich for stromal cells. Cells isolated from the Percoll-light fraction, between the 1.065 and 1.0 layers, were then resuspended in 0.5% BSA (Sigma-Aldrich), 2 mM EDTA (TekNova) (FACS buffer) and counted. To prepare the cells for droplet-based sequencing, mTECs were flow-sorted into tubes containing 750ul DMEM + 10%FBS (50,000 cells were sorted for the Aire trace experiment, 60,000 cells were sorted for the week 2 control, 37,000 cells were sorted for the week 2 experiment, 44,000 cells were sorted for the week 4 experiment, 32,000 cells were sorted for the week 6 experiment, 50,000 cells were sorted for the week 10 experiment, and 24,000 cells were sorted for the week 10 control). Cells were spun down at 300g for 5 minutes and all but 50-100ul of supernatant was removed to aim for a final concentration of 700-1000 cells/ul. 10x genomics single-cell isolation and RNA sequencing