Name: GSM6156736
Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: The 11 frozen tissues were thawed on ice and 10 mg of tissue per IP reaction was chopped into ~1 mm3 pieces with two razor blades on the ice. Chopped tissues were washed out with PBS buffer containing Protease K, 10mM PMSF, and 10mM Sodium Butyrate histone deacetylase inhibitor to remove blood from the tissue. Washed tissue was grinded using pestle and mortar. Then prepared cell mass was cross-linked in the PBS buffer with 1.5% Formaldehyde added at room temperature(RT) for 20 minutes. By adding 125 mM Glycine and placing the samples on the rotator for 5 min, cross-linking reactions were stopped. Fixed cell mass was washed twice with PBS buffer containing Protease K, 10mM PMSF, and 10mM Sodium Butyrate. Fixed cell mass was lysed using buffer A (5 mM PIPES buffer, 85 mM KCl, and 0.5% NP 40). The supernatant was centrifuged out and added to buffer B (50 mM Tris-HCl, 0.5% SDS, and 2.5mM EDTA). All buffers contained Protease K, 10 mM PMSF, and 10 mM Sodium Butyrate histone deacetylase inhibitor. Sonication was then carried out to get 300-500 bp size fragments using Bioruptor Pico (Diagenode). Twenty to fifty cycles of 30 seconds, on and off, were performed at 4 ℃ depending on the tissue. Liver cells were sonicated for only 20 cycles. Chromatin solution was centrifuged to remove the debris and diluted with ChIP IP buffer (16.7 mM Tris-HCl, 0.05% SDS, 1.1% Triton X-100, 1.2 mM EDTA, and 167 mM NaCl). After adding 5 µg of sample to 10 µg of anti-H3K4me1 (ab8895), H3K27Ac (ab4729), H3K4me3 (ab8580), H3K27me3 (ab6002), H3K9me3 (ab8898), and IgG (Sc-2027) per IP reaction, the chromatin solution was incubated overnight at 4 ℃. The cross-linking was reversed and DNA was purified after treatment with protease K and RNase A. ChIPed DNA was quantified using the Qubit fluoro-spectrometer (ThermoFisher Scientific, Waltham, MA, USA) and the enrichment was validated by PCR. The ChIP library was prepared using the TruSeq ChIP Library Preparation Kit and sequenced in ~500 bp fragments using HiSeq 2500 system (Illumina) by Macrogen (Macrogen, Korea).