GSM4185614: ChIP_H3K27ac_tko_1; Mus musculus; ChIP-Seq
About 130 mg of frozen liver was minced in 2 mL of cold 1X DPBS (Thermo Fisher 14080055). The volume of liver samples were brought up to 10 mL of 1X DPBS and 1% formaldehyde. The samples were incubated at room temperature for 8 minutes with rotation. To quench, reaction was brought to 0.125 M glycine and incubated for 5 minutes at room temperature with rotation. Samples were centrifuged at 2500 RCF for 2 minutes at 4°C and the supernatant was discarded and washed once with 1XDPBS. The pellet was resuspended in 1 mL of cold ChIP cell lysis buffer (10 mM Tris- HCl pH 8.0, 10 mM NaCl, 3mM MgCl2, 0.5% IGEPAL CA-630, 1X cOmplete protease inhibitor cocktail) and transferred to a 10 mL tissue grinder on ice. The samples were homogenized with a smooth teflon pestle 20 times and incubated at 4°C for 5 minutes. Following incubation, the samples were centrifuged for 5 minutes at 17,000 RCF at 4°C to pellet nuclei. The supernatant was discarded, and nuclei were resuspended in 1 mL of cold ChIP nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 5 mM EDTA, 1% SDS, 1X cOmplete protease inhibitor cocktail). The samples were sonicated for two rounds using the Standard Biruptor (diagenode UCD-200) with the following parameters: Intensity = High (H), Multitimer = OFF: 30 sec, ON: 30 sec, Total timer = 7.5 minutes with maintaining 4C tempertures. Biruptor was cooled down for 15 minutes between rounds. Following sonication, the samples were centrifuged at 17,000 RCF for 10 minutes at 4°C and the supernatant containing sheared chromatin was recovered. To determine the amount of material required for precipitation we isolated DNA from sonicated chromatin. DNA was quantified using NanoDrop-1000 (Thermo Fisher) and ran on a BioAnalyzer High Sensitivity Chip. Following quantification, 10 μg of sonicated chromatin was added to 1 mL of ChIp dilution buffer (16. mM Tris-HCl pH 8.0, 167 mM NaCl, 0.01% SDS, 1.1% Triton-X 100, 1X cOmplete protease inhibitor cocktail). 3 μg of the following antibodies, H3K4me1(Abcam ab8895), H3K27ac (Active Motif 39133) and HNF4a (Abcam ab181604) were added to the samples and the samples were incubated at 4°C overnight with rotation. 40 μL of recombinant protein G agarose beads (Thermo Fisher) were washed three times with 1 mL of ChIP dilution buffer by resuspending beads in the buffer. Following the washes, the beads were resuspended in 75 μL of ChIP Dilution Buffer and 5 μL of BSA (New England BioLabs) and incubated at 4°C overnight with rotation for blocking. Then chromatin samples were added to the blocked beads and the samples were incubated at 4°C for 1 hour with rotation. Samples were centrifuged at 100 RCF for 30 seconds and the supernatant was discarded. The beads were then washed sequentially with each of the following buffers by resuspending the beads in 1 mL of buffer, incubating beads at room temperature for 5 minutes with rotation, pelleting beads, and discarding supernatant: TSE I (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), TSE II ( 20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, 0.1% SDS, 1% Triton X-100), ChIP Buffer III (10 mM Tris-HCl pH 8.0, 0.25 M LiCl, 1 mM EDTA, 1% IGEPAL CA-630, 1% Sodium Deoxycholate), TE (10 mM Tris-HCl pH 8.0, 1 mM EDTA). Following the washes, the beads were resuspended in 100 μL of elution buffer (0.1 M NaHCO3, 1% SDS) and incubated at room temperature for 15 minutes with rotation. 4 μL of 5M NaCl was added to the supernatant and then samples were incubated at 65°C overnight. Next, DNA was isolated from the samples. Enrichment of IP samples was measured through qPCR using the following program: 95°C for 3 minutes, for 40 cycles, 95°C for 5 seconds, 60°C for 20 seconds, with the specific primers. Then, Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina.  Libraries were sequenced on the Illumina Hiseq 4000.