title: GSM3317131: ZT16 mouse1; Mus musculus; ChIP-Seq
library_construction_protocol: ~ 50 mg of blood perfused liver tissue were dissected from C57BL/6J mice, snap frozen in liquid nitrogen and stored at -80°C. Frozen liver tissues were minced in ice-cold PBS using razor blades into 1 mm cubed, and then homogenized by pushing through 1.5-inch 18G needle for 10 times and then 1.5-inch 21G needle for 20 times. Homogenized cells were immediately crosslinked with 1% formaldehyde for 10 min. The crosslink was quenched by adding glycine to a final concentration of 125 μM. After two washes with cold PBS, the nuclei were lysed in 150 μl nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA) containing 1x EDTA-free protease inhibitor cocktail (Roche), by keeping on ice for 5 min. Immediately, to fragment the chromatin, the lysates were sonicated 17 times for 30 sec by using Bioruptor (Diagenode). After centrifuged at 17,000 rpm for 15 minutes at 4°C, the 150 μl fragmented chromatin was diluted 10-fold with IP dilution buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA) supplemented with 1x EDTA-free protease inhibitor cocktail, and then incubated with 7 μg anti-HNF4A (abcam cat. ab41898) overnight at 4°C. 100 μl of Dynabeads Protein G (Invitrogen cat. #10004D) was added to the chromatin and incubated for another 3 h. Afterwards, the beads were washed once with low salt wash buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA) and once with high salt wash buffer (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA). 200 μl of IP elution buffer (1% SDS, 100 mM NaHCO3) was applied to the bead pellet and incubated at 30°C for 15 min. The eluate was added with 5M NaCl to final conc. of 200 mM and reverse-crosslinked at 65°C overnight. To remove RNA and protein, RNase A and proteinase K were subsequently applied by incubating at 45°C for 1 h. Finally, we diluted the DNA with 5 volumes of Qiagen buffer PB (QIAquick PCR Purification Kit) and purified with QIAquick PCR Purification columns. We eventually resolved our DNA fragments in 30 μl Qiagen Buffer EB. For library preparation, 2 ng of each sample was prepared using the NEB Ultra DNA Library Prep Kit for Illumina following manufacturer's instructions. Each library was dual size selected with 0.55X and 0.14X Ampure beads followed by PCR amplification with Kappa HiFi 2x PCR mix (15 cycles). PCR product was then quantitated using the Qubit (Thermo Fisher) and BioAnalyzer (Agilent BioAnalyzer 2100) to determine library quantity. Libraries were then gel purified on a 2% agarose gel to remove secondary PCR amplification artifacts, quantitated on the Qubit and loaded onto a NextSeq500 flowcell for 1 x 76 base single-end sequencing to generate approximately 20M reads per sample.
attributes.tag: GEO Accession
attributes.value: GSM3317131