GSM3554253: H3K9ME3_HSV-1_2H_ChIPSeq_rep3; Human alphaherpesvirus 1; ChIP-Seq
Briefly, approximately 1×10^7 cells were fixed with 1% formaldehyde for 10 min and quenched by glysine. The cells were washed three times with PBS, then harvested in ChIP lysis buffer (50 mM Tris-HCl, pH7.6, 1 mM CaCl2, 0.2% Triton X-100) and incubated for 5 min with gently rotation. After centrifugation at 12000 rpm at 4℃ for 2 min, lysates were washed once by digestion buffer (50 mM Tris-HCl pH 8.0, 1 mM CaCl2, 0.2% Triton X-100). Then lysates were incubated in 630 μL digestion buffer with 1 μL MNase (NEB, M0247S) at 37℃ for 20 min and then quenched with 8 μL 0.5 M EDTA. Whole lysates were sonicated for 5 min (0.5 s on / 0.5 s off, 25% power) and the supernatants were taken out after centrifugation at 12000 rpm, 4℃ for 10 min. Immunopreciptation was performed with 150 μL sheared chromatin, 2 μg antibody, 50 μL Protein G sepharose 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℃. The immunocomplexes were washed once each with Wash buffer I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), Wash buffer II (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS) and Wash buffer III (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 1% Na-deoxycholate, 1% NP-40), and twice with TE (10mM Tris-HCl pH 8.0, 1mM EDTA). The bound materials were eluted twice with 100 μL elution buffer (1% SDS, 0.1M NaHCO3) and 1μL Proteinase K (20 mg/mL) at room temperature for 10 min. Eluted materials were incubated at 65℃ for 6 hr and then purified with DNA purification kit (TIANGEN DP214-03). ChIP-seq libraries were constructed by VATHS Universal DNA Library Prep Kit for Illumina (Vazyme ND604). Briefly, 50 μL purified ChIP DNA (8-10 ng) was end-reparied for dA tailing, followed by adaptor ligation. Each adaptor marked with a barcode of 6 bp which can be recognized after mixing different sample together. Adaptor-ligated ChIP DNA was purified by AMPure XP beads (1:1) and then amplifying by PCR of 11-13 cycles with the primer matching with adaptor universal part. Amplified ChIP DNA was purified again using AMPure XP beads (1:1) in 35 μL EB elution buffer. For multiplexing, libraries with different barcode were mixed together with equal molar quantities by considering appropriate sequencing depth (30-40 million reads per library). Libraries were sequenced by Illumina Hiseq X Ten platform with pair-end reads of 150 bp.
GEO Accession
GSM3554253