GSM5380841: week-2_H3K4me3_rep2; Mus musculus; ChIP-Seq
DNA were clarified from nuclei treated with micrococcal nuclease and histone-DNA complexes were isolated with antibody. Briefly, around 30 milligrams of each tissue were cut into 1 mm3 pieces in PBS with protease inhibitor. Tissue pieces were cross-linked for 10min at room temperature with 1% formaldehyde and then quenched with 0.125 M of glycine for 5 min. Cross-linked tissues were triturated in tissue grinding instrument for 60s at 60 Hz, and then centrifuged at 12000 rpm, 4 °C for 10 min. Supernatant with massive oil was discarded and the precipitates were lysed with 1 mL lysis buffer (50 mM Tris-HCl pH 8.0, 0.1% SDS, 5 mM EDTA) for 4 min with gentle rotation. After centrifugation at 12000 rpm, 4 °C for 2 min, the pellet was washed once with digestion buffer (50 mM Tris-HCl pH8.0, 1 mM CaCl2, 0.2% Triton X-100), incubated in 630-μL digestion buffer with 1 μL MNase (NEB, M0247S) at 37°C for 20min and quenched with 8 μL 0.5M EDTA. The resulted mixture was sonicated and the pellet was discarded after centrifugation. 30 μL supernatant was taken for checking the efficiency of digestion. Immunoprecipitation was performed with 150 μL sheared chromatin, 2 μg antibody, 50 μL Protein G beads and 800 μL dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS) overnight at 4 °C. Next day, the beads were washed once with Wash buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with Wash buffer II (20mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), once with Wash buffer III (10mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Na-deoxycholate, 1% NP-40) and twice with TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). The beads were eluted twice with 100 μL elution buffer (1% SDS, 0.1M NaHCO3, 20mg/mL Proteinase K) at room temperature. The elution was incubated at 65 °C for 6 h and then purified with DNA purification kit (TIANGEN DP214-03). ChIP-seq libraries were constructed with ChIP and input DNA using VATHS Universal DNA Library Prep Kit for Illumina (Vazyme ND606). Briefly, 10 ng DNA was end-repaired for dA tailing, followed by adaptor ligation. Each adaptor was marked with a barcode of 8 bp DNA. Adaptor-ligated DNA was purified by AMPure XP beads (1:1) and then amplified by PCR of 10 cycles with the primer matching with adaptor universal part. Amplified DNA was purified again using AMPure XP beads (1:1) in 35 μL EB elution buffer. For multiplexing, libraries with different barcode were mixed with equal molar quantities (30-50 million reads per library). Libraries were sequenced by Illumina Nova-seq platform with pair-end reads of 150 bp.