GSM4783819: C57_H3K27ac_rep2; Mus musculus; ChIP-Seq
Cultures were harvested and cells were washed in PBS prior to downstream analysis. RNA was harvested using Trizol followed by purification with the Zymo MicroRNA kit (R2061). ChIP lysates were generated from sonicated nuclei and histone-DNA complexes were isolated with antibody. ATAC-seq: A total of 6.5x104 viable eosinophils were pelleted at 500 g for 5 minutes at 4 C prior to resuspension in 50 uL ATAC-Resuspension Buffer (ATAC-RSB; 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2 made up to 50 mL in ddH2O) with added 0.1% NP-40, 0.1% Tween-20 and 0.01% Digitonin. Samples were incubated on ice for 3 minutes prior to washing in 1 mL cold ATAC-RSB containing 0.1% Tween-20 only. Nuclei were pelleted at 500 g for 10 minutes at 4 C prior to addition of 50 uL transposition mixture (2X TD buffer, 100 nM transposase enzyme, 5 uL ddH2O, 1% Digitonin, 10% Tween-20 made up to 50 mL in 1X PBS) and incubated at 37 C for 30 minutes. After tagmentation, the transposased DNA was purified using the MiniElute Reaction Cleanup Kit (28204, Qiagen). RNA libraries were prepared for sequencing according to the TruSeq Stranded mRNA kit protcol. ChIP libraries were constructed by ChIPmentation. ATAC libraries were constructed with the OMNI-ATAC protocol.