H3K27ac HiChIP Protocol for Chromatin Interactions

We followed the HiChIP protocol published by Mumbach et al.^{37 (link)}, using antibody to H3K27ac (Abcam, ab4729) with the following modifications. The EBs were first treated with StemPro Accutase Cell Dissociation Reagent (Thermo Fisher) at 37 °C for 10–15 min with pipetting. Approximately one million cells were crosslinked with freshly prepared 1% formaldehyde. The pellet was then resuspended in 500 μl of ice-cold Hi-C Lysis buffer. After digestion with 25 U (5 μl of 5U/μl) MboI restriction enzyme and ligation, the nuclear pellet was brought up to 880 μl of Nuclear Lysis Buffer. Samples were sheared using a Covaris E220 using the following parameters: fill level = 10, duty cycle = 5, PIP = 140, cycles/burst = 200, time = 2 min and then clarified by centrifugation for 15 min at 16,100 × g at 4 °C. The samples were precleared with 6 μl Dynabeads Protein A (Thermo Fisher) at 4 °C for 1 h. We then added 2.5 μg of antibody to H3K27ac, and captured the chromatin-antibody complex with 6 μl of Dynabeads Protein A. Approximately 2–4 ng of ChIP DNA was obtained following Qubit quantification. The amount of Tn5 used and number of PCR cycles performed were based on the post-ChIP Qubit amounts, as described in the HiChIP protocol^{37 (link)}. The library was sequenced on Illumina NextSeq 500 with 75 bp paired-end reads. Total 13 million cells were used in HiChIP experiment.