GSM6481393: MD6, H3K27ac, HiChIP; Homo sapiens; OTHER
HiChIP was performed as described (Mumbach et al., 2016) with some modifications. Briefly, ChIP protocol was replaced with the one reported in Crispatzu et al., 2021, using H3K27ac antibody (Abcam #ab4729), starting from 1*10 6 cells per condition. Digestion and ligation were performed overnight (NEB T4 Ligase, #M0202 instead of Invitrogen T4, 15224-041) and DNA was extracted with phenol-chlorophorm. After biotin pull-down, beads were resuspended in 25 μL of 2X TD Buffer, 0.5uL of Tn5 (Illumina #1503861), and water to 50 μL. After transposition, beads were resuspended in 50 µL of Phusion HF 2× (New England Biosciences). Generally, 14 cycles were used for Tn5 Nextera PCR amplification (Illumina Nextera DNA UD Indexes Kit) with the following PCR program: 72 °C for 5 min, 98 °C for 1 min, then cycle at 98 °C for 15 s, 63 °C for 30 s, and 72 °C for 1 min. 50ul of PCR product were size-selected with 25ul Ampure XP Beads and separated on a magnet. The supernatant was incubated again with 90ul Ampure XP Beads. The supernatant was then discarded, beads were combined, washed with Ethanol and eluted in 30ul of Elution buffer. 1ul was loaded onto D1000 Screen Tape (TapeStation) for a second size check, where library size and molarities were extracted. Then, qPCR was performed with standard Illumina primers in order to quantify the libraries. Finally, samples were pooled with different Index/Adapter sequences and sequenced on a NovaSeq 6000