Nebnext q5 master mix

HiChIP was performed as described by Mumbach et al.^{71 (link)}. with the following modifications. Per condition, three biological replicates were generated. 5 × 10⁶ K562 dTAG-BRD4 cells^{47 (link)} were treated with 500 nM dTAG7 or DMSO (control) for 2 h, followed by crosslinking with 1% methanol-free formaldehyde for 10 min at room temperature. The partially lysed nuclei were incubated with 375 U MboI (NEB, Cat.# R0147M) at 37 °C overnight. After biotin-dATP incorporation, chromatin shearing was conducted in a Covaris S220 sonicator for 6 min at intensity 4, duty cycle 5% and 200 cycles per burst. For IP, 5 µg of an H3K27ac-specific antibody (Abcam, ab4729) coupled to Pierce Protein A/G (Thermo Fisher Scientific, Cat.# 10004D) were used per sample. The eluted DNA was heated to 65 °C for 5 h, treated with 80 µg RNase A (Thermo Fisher Scientific, Cat.# 10753721) for 2 h at 37 °C and with 200 µg Proteinase K (Carl Roth, Cat.# 3719.2) for 45 min at 50 °C. 50 ng of the purified DNA was incubated with 2.5 µl of Tn5 (Illumina, Cat.# 20034197). NEBNext Q5 master mix (NEB, Cat.# M0544S) was used to amplify the library by 8 cycles. Lastly, the libraries were size-selected using AMPure XP beads (Backman Coulter, Cat.# A63881) at a ratio of 0.8 and subsequently 0.5 to enrich fragments of 200–750 bp and sequenced in PE75 or PE100 mode on an Illumina HiSeq 4000 or NovaSeq 6000 sequencer.