Instrument: Illumina NovaSeq 6000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: 100 embryos at dome stage were collected for each sample and fixed in cross-linking buffer (50mM HEPES pH8.0, 0.1 M NaCl, 1 mM EDTA, 0.5 M EGTA, 1 % Formaldehyde) for 15 min at room temperature. For chromatin fragmentation, 400 μl ChIP lysis buffer (50 mM Tris, pH 8.0, 10 mM EDTA, 0.1 % Sodium Deoxycholate, 1% Triton-X) with 1 × PIC and 1 mM PMSF were added to fixed embryos and incubated in ice for 20 min. Then, sonication was performed on ME220 with 10% duty factor, 75 peak incident power, 1000 cycles/burst, and for 600 second per tube. Fragmented chromatins were centrifuged at 12,000 rpm for 10 min at 4°C to remove cellular debris. For IP, 2 μg RNA Pol II antibody (Active Motif, no. 61667) and 10 μl Dynabeads™ Protein G were added to the fragmented chromatin solution and incubated at 4°C overnight with rotator at speed of 10 rpm. Beads containing immuno-bound chromatin were collected by placing the microfuge tube on a magnet rack and washed with ChIP lysis buffe 2 times, High salt wash buffer (10 mM Tris, pH 8.0, 500 mM NaCl, 1 mM EDTA, 1 % Triton-X, 0.1 % SDS, and 0.1% Sodium Deoxycholate) 2 times, LiCl wash buffer (10 mM Tris, pH 8.0, 250mM LiCl, 1 mM EDTA, 1 % Triton-X, 0.5 % SDS, and 0.5% Sodium Deoxycholate) 1 time, and TE 2 times, mixing 5 minutes for each wash on a rotator at 4°C. Then, the beads were suspended in 200 μl IP Elution Buffer with 200 μg/ml Proteinase K and incubated at 65°C overnight for reverse cross-linking, following DNA extraction with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1, pH 7.8) with phase-lock tubes. The supernatant containing ChIP DNA was precipitated by addition of an equal volume of isopropanol and 1 μl GlycoBlue (Thermo Fisher) for 12 h at -20°C. Accel-NGS 1S Plus DNA Library Kit was used for library construction following the manual.