GSM8615551: d3a-ko-r1; Mus musculus; ATAC-seq
Murine pulmonary EC were subjected to FACS, pelleted for 7 min at 500 × g in a prechilled (4°C) fixed-angle centrifuge, and resuspended in 50 µl of cold lysis buffer (10 mM Tris-HCL (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.1% NP40, 0.1% Tween20, 0.01% Digitonin). The nuclei isolation reaction was incubated for 3 min at room temperature. After lysis, 1 ml of ATAC-RSB (10 mM Tris-HCL (pH 7.4), 10 mM NaCl, and 3 mM MgCl2) containing 0.1% Tween20 was added and mixed. Nuclei were pelleted for 10 min at 500 × g at 4°C and resuspended in 50 µl of transposition buffer (10 mM Tris-HCl (pH 7.6), 5 mM MgCl2, 10% Dimethyl Formamide, 2.5µl Transposase (100nM final concentration), 0.1% Tween20, 0.01% Digitonin,16.5µl PBS, 5µl H2O). Transposition reaction was incubated for 30 min at 37°C in a thermomixer shaking at 1000r.p.m. The reaction was stopped with 20 µl of 5 M guanidinium thiocyanate, the ATAC raw material was purified with a 2x volume of Ampure XP DNA purification beads, and the DNA was eluted in 25 µl of H2O. The DNA eluate was PCR amplified using 1x NEBNext High Fidelity Mix in a total volume of 50 µl. The number of cycles used for library amplification depended on the observed amplification curve and varied between 10 and 15 cycles. ATAC libraries were purified with a 1.4x volume of Ampure XP DNA purification beads, and the DNA was eluted in 12 µl of elution buffer.