Instrument: Illumina Genome Analyzer
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: All the tissues were ground in the liquid nitrogen and fixed by 1% formaldehyde at room temperature for 15 min. 0.2 M glycine was added and incubated at room temperature for 5 min to quench the fixation. Fixed tissues were then washed once by cold 1X PBS. Tissue pellet was resuspended and incubated on ice for 10 min in 100 µL of ChIP-seq lysis buffer (20 mM Tris-HCl, pH 8.0, 1% SDS, 50 mM EDTA, 1X proteinase inhibitor cocktail). Next, 900 µL cold 1X TE buffer was added to dilute SDS concentration and the nuclei suspension was sonicated using Covaris E220 with the following parameters: 140 W, duty factor 5, 200 per burst. Sonication time is variable, depending on tissue types. To check the chromatin fragmentation size, 20 µL of input chromatin was reverse crosslinked in elution buffer (20 mM Tris-HCl, pH 8.0, 1% SDS, 1 mM EDTA) at 65 °C overnight, treated with Rnase A and proteinase K and purified by phenol-chloroform extraction. Input DNA was then loaded on a Lonza flash gel to make sure that the majority of DNA size distributes between 100-300 bp. To prepare antibody-beads complex, 3 µg of histone H3K27ac antibody (Active Motif, 39133) or H3K4me3 antibody (EMD Millipore, 07-473) was mix with 12 µL M-280 sheep anti-rabbit IgG Dynabeads (ThermoFisher, 11203D) in 150 µL of 5 mg/mL BSA/1X PBS buffer, with rotation at 4 °C for 3 h. After incubation, antibody-beads complex was washed once with BSA/1X PBS buffer. About 200 µg chromatin was used per IP. Equal volume of master mix (1X TE, 2% Triton X-100, 0.2% sodium deoxycholate, 2X proteinase inhibitor cocktail) was mixed with 200 µg chromatin and then incubated with antibody-beads complex overnight with rotation. The next morning, the beads were washed 5 times with cold RIPA wash buffer (20 mM Tris-HCl, pH 8.0, 1% NP-40, 0.7% sodium deoxycholate, 500 mM LiCl, 1 mM EDTA, 1X proteinase inhibitor cocktail). Then the chromatin bound on the beads was eluted by 150 µL of elution buffer at 65 °C for 30 min. To prepare the library, eluted chromatin was reverse crosslinked and purified by phenol-chloroform extraction. Then DNA was end-repaired by END-IT DNA end-repair kit (Epicentre, ER81050) according to kit's protocol, added A using Klenow fragment (3'->5' exo-) (NEB, M0212S), ligated with Illumina TruSeq adaptor (Illumina, FC-121-3001) and subsequently amplified by PCR (Roche, kk2601). The quality and quantity of all the libraries were checked using BioAnalyzer High Sensitivity DNA Kit (Agilent). The libraries were sequenced on Illumina HiSeq 2500 with reads length of 2X 50 bp or 2X 100 bp.