title: GSM5260484: ChIP Input on FoxA Dev double null - sample 2; Mus musculus; ChIP-Seq
library_construction_protocol: 130 mg of frozen liver was minced in 2 mL of cold 1X DPBS (Thermo Fisher 14080055). The volumes of liver slices were brought up to 10 mL of 1X DPBS and 1% formaldehyde and incubated at room temperature for 8 minutes with rotation. Reaction was brought to 0.125 M glycine for quenching and incubated for 5 minutes at room temperature with rotation. Samples were centrifuged at 2,500 RCF for 2 minutes at 4°C, the supernatants discarded, and pellets washed with 1X DPBS. 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 on ice to a 10 mL tissue grinder. The samples were homogenized 20 times with a smooth Teflon pestle 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 the nuclei. After 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). Then, we performed two rounds of sonication 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 4°C. Following sonication, samples were centrifuged at 17,000 RCF for 10 minutes at 4°C and supernatant was recovered. We isolated DNA from the sonicated chromatin to determine the amount of material required for precipitation. The DNA concentration was quantified using a NanoDrop-1000 (Thermo Fisher) and run on a BioAnalyzer High Sensitivity ChIP to estimate the length distribution. Then, 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, 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. Next, 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 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). After 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 using Minielute (Quiagen). 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. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina. Libraries were sequenced on the Illumina Hiseq 4000 with 50 bp single-end sequencing with a minimum of 20 million reads per sample.