GSM1318182: FOXA2_ChIPSeq_Embryonic_Liver; Mus musculus; ChIP-Seq
Cells were homogenized in 1% formaldehyde and incubated at room temperature (RT) for 10 min, prior to the addition of glycine to 0.125 M followed by incubation at RT for 5 min. Cells were pelleted, washed and resuspended in 5 volumes ChIP cell lysis buffer (10 mM Tris–Cl, pH 8.0, 10 mM NaCl, 3 mM MgCl2, 0.5% NP-40) containing protease inhibitor cocktail tablets (Roche, Laval, QC, Canada). Cells were then rehomogenized, incubated on ice for 5 min and repelleted. The pellet was resuspended in 3 volumes ChIP nuclear lysis buffer (1% SDS, 5 mM EDTA, 50 mM Tris–Cl, pH 8.1) supplemented with protease inhibitor cocktail tablets (Roche). Cells were sonicated on ice-water (Sonicator 3000, Misonix, Farmingdale, NY, USA) for 20 cycles of 30 s on, 40 s off and 60 μg of chromatin was precleared with Protein G beads (100 μl, Active Motif), Protein Inhibitor Cocktail (0.5 μl, Active Motif) and ChIP dilution buffer (to 250 μl, 0.01% SDS, 1.1% Triton X-100, 167 mM NaCl, 16.7 mM Tris–Cl, pH 8.1). Beads were precipitated and 3 μg of Foxa2 [HNF-3β (M-20): sc-6554] or HNF4a [sc-8987] antibody was added to supernatants. Fresh Protein G beads were also blocked with 1 mg/mg BSA and 0.1 mg/mg herring sperm DNA in ChIP dilution buffer. Following overnight incubation, rocking at 4°C, the samples were incubated with the blocked beads for 4 h rocking at 4°C. The beads were then precipitated and washed in low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–Cl, pH 8.1, 150 mM NaCl), high salt buffer (low salt buffer with 500 mM NaCl), lithium chloride buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris–Cl, pH 8.1) and twice with TE buffer. Bead complexes were eluted twice by addition of 125 μl elution buffer (1% SDS, 0.1 M NHCO3) and rotation for 15 min at RT. NaCl was added to 0.192 M to reverse crosslink and samples were incubated overnight at 65°C. Samples were then incubated with Proteinase K (Invitrogen, Carlsbad, CA, USA) and RNaseA (Sigma-Aldrich, Oakville, ON, Canada) for 2 h at 50°C. DNA was purified by two rounds of phenol–chloroform extraction and ethanol precipitation and resuspended in 50 μl dH2O. ChIP DNA was run and size separated by PAGE (8%). The 100-300bp DNA fraction was excised and eluted from the gel slice overnight at 4°C in 300 μl of elution buffer (5:1, LoTE buffer (3 mM Tris-HCl, pH 7.5, 0.2 mM EDTA)-7.5 M ammonium acetate), and was purified using a Spin-X Filter Tube (Fisher Scientific), and by ethanol precipitation. The library was prepared by following the pair end library protocol (Illunima Inc., USA). Briefly, the DNA was subject to end-repair, and phosphorylation by T4 DNA polymerase, Klenow DNA Polymerase, and T4 polynucleotide kinase respectively in a single reaction, and then 3’ A overhangs generation by Klenow fragment (3’ to 5’ exo minus), and ligated to Illumina PE adapters, which contain 5’ T overhangs. The adapter-ligated products were purified on Qiaquick spin columns (Qiagen), then PCR-amplified with Phusion DNA Polymerase in 10-15 cycles using Illumina’s PE primer set (Illumina). PCR product of desired size range was purified using a 8% PAGE, and the DNA quality was assessed and quantified using an Agilent DNA 1000 series II assay and Nanodrop 7500 spectrophotometer (Nanodrop, USA) and diluted to 10nM. The final concentration was double checked and determined by Quant-iT dsDNA HS Assay Kit using Qubit fluorometer (Invitrogen). Clusters were generated on the Illumina cluster station and sequence was run on Illumina Genome Analyzer following the manufacturer’s instructions.