Instrument: Illumina HiSeq X Ten
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Cells were crosslinked by 1% formaldehyde for 10 mins, and then quenched in 125 mM glycine for 5mins. After washing with cold TBS twice, cells were incubated in lysis buffer (10 mM Tris-HCl, pH7.5, 10 mM NaCl, 0.5% NP-40) for 10 mins on ice. Nuclei were washed and resuspended in MNase digestion buffer (20 mM Tris-HCl, pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl2) supplemented with 1,000 units of MNase (NEB) and incubated at 37 °C with continuous mixing for 20 mins. The reaction was stopped by adding of 2XSTOP buffer (100 mM Tris-HCl, pH8.1, 20 mM EDTA, 200 mM NaCl, 2% Triton X-100, 0.2% sodium deoxycholate), followed by sonication for 10 mins (30 secs on / 30 secs off). For spike-in ChIP-seq, the MNase digested chromatin from drosophila S2 cells was added to 1% to 5% of total chromatin. The chromatin was incubated with antibodies at 4 °C overnight and then subjected to 30 μl of protein G-magnetic beads for additional 2 hours. The beads were extensively washed and bound DNA was eluted with elution buffer (10 mM Tris-HCl, pH8.0, 10 mM EDTA, 150 mM NaCl, 5 mM DTT, 1% SDS) and reverse-crosslinked at 65 °C overnight. DNAs were purified using Min-Elute PCR purification kit (Qiagen) after treatment of proteinase K and RNase A. Sequencing libraries were prepared using TruPrep DNA library prep kit (Vazyme) following the manufacturer's instructions. The library was sequenced on an Illumina HiSeq Xten platform with pair-end read of 150bp