title: GSM8679701: CTCF_Caco-2_rep2; Homo sapiens; OTHER
library_construction_protocol: For CUT&RUN-seq, cells are incubated with activated concanavalin A beads and resuspended with buffer containing 0,06 % digitonin. For CUT&RUN-seq, specific antibodies are added, H3K27ac (Ab4729), CTCF (Active Motif 61311) and incubated at 4°C for 2 hours. pAG-MNase is activated for 30 minutes on ice.  Stop buffer is added and samples are incubated at 37°C for 30 minutes to release DNA fragments. End-repair and A-tailing are then performed using the KAPA HyperPrep kit (Roche). Size selection is performed using AMPure XP beads (Beckman Coulter) with double purification by addition of HXP buffer (20% PEG 8000, 2.5 M NaCl). Amplified libraries are then obtained by adding HotStar ReadyMix 2X and primer mix 10X. Libraries are amplified using NEBNext High-Fidelity 2 × PCR Master Mix (New England Biolabs). PCR products are purified using QIAquick PCR purification kit (Qiagen). Libraries are paired-end sequenced using Illumina sequencing (NextSeq500 Illumina).