title: GSM8951303: CNR_501mel_set1_dCas9_ALK_sgCtrl1_H3K27ac; Homo sapiens; OTHER
library_construction_protocol: CUT&RUN was performed using CUT&RUN assay kit (Cell signaling technology). Briefly, 0.25 x 106 live cells were immobilized on activated Concanavalin A beads and permeabilized with digitonin. Cell:bead suspension was washed with 1x wash buffer supplemented with spermidine and protease inhibitor cocktail and incubated with 1 µg of antibody at 4 °C overnight. The following antibodies were used: anti-H3K27ac (Abcam, ab4729) and anti-H3K9me3 (Abcam, ab176916) antibodies. Antibody-targeted digestion of chromatin was performed by incubation of pAG-MNase at 4 °C for 1 hr and activation by calcium chloride at 4 °C for 30 min. MNase cleavage was stopped by addition of 1x stop buffer. Digested chromatin was allowed to diffuse out of cells at 37 °C for 20 min and DNA was purified by Nucleospin gel and PCR clean-up spin column (Takara bio). Sequencing libraries were prepared using the KAPA HTP Library Preparation Kit (KAPA Biosystems). Barcoded libraries were run on NovaSeq 6000 in a PE100 run. An average of 25 M reads were generated per sample.