title: GSM8217637: Stroma_1mo_ZZ_2; Mus musculus; RNA-Seq
library_construction_protocol: The thymus from three 1-month-old ZZ homozygotes and wild-type littermates were collected and pooled per single-cell dataset. Connective tissues, excess blood vessels, and adipocytes were carefully removed in 4'C sterile 1X DPBS. Clean thymuses were then transferred to digestion buffer (2% FBS/1640 RPMI (w/ 25mM HEPES), DNase I (20µg/µL), Collagenase/Dispase (1mg/mL)) to be thinly sliced into 1~2mm fragments. The digestion solution containing the thymic fragments was then incubated at 37'C for 1 hour. In between the incubation period, the solution was gently mixed up and down with transfer pipettes every 20 minutes, 100 times each. Post-digestion, the solution was passed through a 70µm cell strainer to remove undigested fragments. Cells were centrifuged at 300 x g, 4'C for 10 minutes. Red blood cells were then removed with a 2-minute incubation in RBC Lysis Buffer (10X) (BioLegend 420301). The solution was centrifuged again at 300 x g, 4'C for 10 minutes. Cells were then resuspended in 3mL MACS buffer (filtered 0.5% BSA/2mM EDTA/1X DPBS). As per MACS separation guidelines, cells underwent CD45 separation with CD45 microbeads, mouse (Miltenyi Biotec 130-052-301). Spleens were freshly collected and gently meshed through 70µm cell strainer. After RBC removal, single-cell suspension was made before sample submission. Library was constructed according to the manufacturer's instructions (single cell 3', 10X genomics).