GSM6304187: mTEClo, WT, ATAC, rep1; Mus musculus; ATAC-seq
Mouse thymus was enzymatically digested and then DAPI-CD45-EpCAM+Ly51-MHCII-high or MHCII-low cells were sorted by FACSAria into 100 uL of BAMBANKER (GC LYMPHOTEC) and kept frozen until library constructions. ATAC-seq libraries were constructed as previously reported by Corces et al. (Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat. Genet. 2016). Specifically, frozen samples were thawed, split into two tubes, and washed with 1 ml of ice-cold PBS containing protease inhibitors (cOmplete ULTRA, EDTA-free; Merck). Cell pellets were resuspended in 35 uL of Tn5 transposase mixture (10 mM TAPS-NaOH pH 8.5, 5 mM MgCl2, 10% DMF, 0.2 mg/ml digitonin, and in-house Tn5) on ice. Then, the cells were incubated at 37C for 30 min with agitation followed by DNA isolation using the Monarch PCR & DNA Cleanup Kit (New England Biolabs) according to the manufacturer's instructions. The tagmented DNA was amplified using KAPA HiFi DNA Polymerase (Roche) with a set of primers designed by Buenrostro et al. (Single-cell chromatin accessibility reveals principles of regulatory variation. Nature 2015) with the following thermal cycles: 5 min at 72C, 30 s at 95C followed by 17 cycles (98C for 20 s, 63C for 10 s), and a final extension at 72C for 3 min. The PCR products were purified using a 1.8x volume of AMPure XP beads (Beckman Coulter) and eluted in EB (Qiagen).