title: GSM9205633: Z2KO_D3_ChIP_H3K4me1_rep1; Mus musculus; ChIP-Seq
library_construction_protocol: For TF ChIPs (ZIC2, ZIC3), one confluent 10 cm plate (~40–50 × 106 cells) was used; for histone mark ChIPs, 1/5 of a 10 cm plate was used (~10 × 106 cells). Cells were cross-linked directly on the tissue culture plate in 1% formaldehyde (Sigma-Aldrich, 252549-100ML) for 10 min at room temperature, followed by quenching with 0.125M Glycine (Sigma-Aldrich, G8898-1KG) during 5–10 min. After two washes with 1X PBS supplemented with cOmpleteª Protease Inhibitor ULTRA Tablets (SigmaAldrich, 5892791001), cell pellets were snap-frozen and stored at –80°C prior to use. Chromatin was extracted by sequential re-suspension in three lysis buffers (Lysis Buffer 1: 50 mM Hepes (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.2% TX-100, dH20 ;Lysis Buffer 2: 10 mM Tris pH 8, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, dH20; Lysis Buffer 3: 10 mM Tris pH 8, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine, dH20), followed by centrifugation (5 min, 1350 g, 4 °C) between steps. Chromatin was sonicated using an EpiShear Probe Sonicator (Active Motif, 53052) under the following conditions: • Histone marks: 30% amplitude, 20 sec on / 30 sec off, 10 min total. • Transcription factors: 35% amplitude, 20 sec on / 30 sec off, 4 min 40 sec total. These conditions aimed to obtain chromatin with sizes 200-500 bp for histone ChIPs and 500-1000 bp for TF ChIPs. After sonication, samples were cleared by centrifugation (10 min, 16000 g, 4 °C), and supernatants were aliquoted. Each aliquot was incubated overnight at 4 °C with 3–5 µg antibody (for histone marks) or 10 µg (for TFs). A 10% aliquot was reserved as input control. The next day, pre-blocked Dynabeads™ Protein G (Thermo Fisher Scientific, 10004D) were added (100 µl for TFs; 75 µl for histone marks) and incubated for 4 h at 4 °C. Beads were washed five times with RIPA buffer (50 mM Hepes pH 7.5, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate, dH2O) and once with TE buffer containing 50 mM NaCl (Sigma-Aldrich, S9888). Chromatin was eluted from the beads by heating at 65°C with occasional vortex. After this, crosslinking was reversed by overnight incubation at 65°C. DNA was purified using DNA Clean & Concentrator kit (Zymo Research, D4034) for inputs and ChIP DNA Clean & Concentrator kit (Zymo Research, D5205) for ChIP samples. Purified DNA concentration was measured with a Qubit 4 Fluorimeter (Thermo Fisher Scientific, Q33238) using the Qubit™ dsDNA HS Assay Kit (Thermo Scientific, Q32854) for ChIP DNA, and the Qubit™ dsDNA BR Assay Kit (Thermo Scientific, Q32853) for input samples. Libraries were prepared for the ChIP and input samples from D0, D3, and D6 by Macrogen Inc. (Korea) using the Illumina TruSeq ChIP-seq protocol. Libraries were sequenced (paired-end 150 bp, 40M reads/sample) on a NovaSeq6000 platform.