Dibo biotin

Hi-ChIP was performed as described (Mumbach et al., 2016 (link)). Briefly, 1 × 10⁷ cells
for each biological replicate were collected and crosslinked by using
1% formaldehyde for 10 minutes. Chromatin was digested using MboI
restriction enzyme (NEB, Cat#R0147) followed by end-repair, ligation
and sonication. Sheared chromatin was cleared and 3-fold diluted as
described in ChIP method and then incubated with anti-H3K27ac antibody at
4°C for overnight. Chromatin-antibody complex was captured by
Dynabead Protein-A bead. Biotin was incorporated by adding DIBO-biotin
(Thermo Fisher, Cat#C-10412) followed by capture with Streptavidin
C-1 bead (Thermo Fisher, Cat#65002). Captured DNA was quantified
using Qubit (Thermo Fisher) and an appropriate amount of Tn5 enzyme was
added to captured DNA to generate sequencing library. Sequencing was
performed on HiSeq 4000 with paired-end read and analyzed by using HiC-pro
(Servant et al., 2015 (link)).

Sample beads were washed in 200 μl of ChIRP elution buffer (150 mM NaCl, 50 mM Tris-HCl, pH 8.0, 3 mM Mgcl₂, 10 mM dithiothreitol, 0.1% NP-40) and then resuspended in 200 μl of ChIRP elution buffer containing 4 μl of RNase A (5 mg ml⁻¹, Thermo) and 4 μl of RNase H (5 U μl⁻¹, Thermo)^{24 (link)}. Samples were then incubated at 37 °C with shaking for 30 min, placed on a magnet and eluted into new tubes. Elution was repeated with another 200 μl of ChIRP elution buffer containing RNases. SDS to a final concentration of 0.5% and 20 μl of Proteinase K (20 mg ml⁻¹, Thermo Fisher) were then added to the 400-μl reaction. Samples were incubated at 55 °C for 45 min with shaking. Samples were purified with DNA Clean and Concentrator columns (Zymo Research) and eluted in 50 μl of water. To incorporate a biotin into the DNA contacts for capture, we carried out copper-free CLICK chemistry using 1 μl of DIBO-Biotin (Thermo) added to the sample and incubating at 37 °C for 1 h with shaking. Samples were purified again with DNA Clean and Concentrator columns (Zymo Research) and eluted in 10 μl of water.