title: GSM9058452: BV2_hyp_ctrl_rep3_H3K27ac; Mus musculus; OTHER
library_name: GSM9058452
library_construction_protocol: CUT&RUN was performed on BV2 cells exposed to hypoxic or normoxic condition, as described previously, using EpiCypher CUTANA ChIC/CUT&RUN Kit (Version 4, EpiCypher, 14-1048), according to the manufacturer's protocol with minor modifications. Briefly, cells were subjected to mild crosslinking with 1% formaldehyde for 3 min, followed by FACS. Then, 5x105 cells per reaction were washed with PBS, and nuclei were isolated using nuclear extraction buffer (20 mM HEPES pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 1 mM MnCl2, 1X cOmplete Mini-Tablet and 0.5 mM spermidine). Activated ConA beads were added directly to the to the nuclear suspensions, followed by overnight incubation at 4°C with H3K27ac (Cell Signaling, 8173S). Chromatin digestion was performed using protein pAG-MNase, and the released DNA fragments were recovered after reverse crosslinking overnight at 55°C in the presence of SDS and proteinase K. Sequencing libraries were prepared with the QIAseq® Ultralow Input Library Kit (Qiagen), according to the manufacturer's instructions.