GSM8278722: MP14WT1; Mus musculus; RNA-Seq
Excess blood vessels, connective tissues, and adipocytes were carefully removed in ice-chilled 4 sterile 1X PBS (w/o Ca2+, Mg2+). Thymi were finely cut into 1~2 mm3 fragments and transferred into digestion buffer (2% FBS/1640 RPMI (w/ 25mM HEPES), DNase I (20µg/µL), Collagenase/Dispase (1mg/mL)). Tubes containing the thymic fragments with digestion buffer were incubated in a 37 water bath for 20 minutes, 3 times. The solution was gently mixed up and down with transfer pipettes in between the incubations, 100 times each. To remove undigested thymic fragments, the solution was passed into a new tube, through a 70µm cell strainer. Single-cell suspension was then centrifuged for 10 minutes at 300rcf, 4, a condition for centrifugation throughout this protocol. After decanting the supernatant, erythrocytes were lysed with a 2-minute-long incubation in 2mL RBC Lysis Buffer (10X) (BioLegend 420301). After another centrifugation & decantation, cells were resuspended in MACS buffer (filtered and degassed 0.5% BSA/2mM EDTA/1X PBS (w/o Ca2+, Mg2+)). Post cell counting with a hemocytometer, cells underwent CD45 separation as per MACS separation guidelines with mouse CD45 microbeads (Miltenyi Biotec 130-052-301) to collect CD45- cells. Library was constructed according to the manufacturer's instructions (single cell 3', 10X genomics).