GSM7717219: Th2cell, cKO2, ATACseq; Mus musculus; ATAC-seq
RNAseq) RNA was extracted using Quick-RNA microPrep kit (Zymo) ﻿ATACseq) Fifty thousand cells were centrifuged at 500 × g for 5 min at 4 °C. Cell pellets were washed once with 1x PBS and cell pellets were resuspended in 50 μl of ATAC-RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP40, 0.1% Tween20 and 0.01% Digitonin, and incubate on ice for 5min. Then, we washed with 1 ml of cold ATAC-RSB containing 0.1% Tween-20 but no NP-40 or digitonin and immediately centrifuged at 500 × g for 10 min at 4 °C. RNAseq) RNA libraries for sequencing were prepared using the NEBNext Ultra II RNA Library Prep Kit following the manufacturer's instructions. ATACseq) The cell pellet was resuspended in the transposase reaction mix (25 μL 2 × TD buffer, 2.5 μL Illumina Tn5 transposase (100 nM final), 16.5 μL PBS, 0.5 ul 1% Digitonin, 0.5 ul 10% Tween-20, and 5 ul H2O). The transposition reaction was carried out for 30 min at 37 °C. Directly following transposition, the sample was purified using a Zymo DNA Clean & Concentrator-5 Kit. Then, we amplified library fragments using NEBNext 2x PCR master mix and 25 uM of PCR primers, using the following PCR conditions: 72 °C for 5 min; 98 °C for 30 s; and thermocycling at 98 °C for 10 s, 63 °C for 30 s, and 72 °C for 1 min.