title: GSM5288542: U2OS_HNF4A_BMAL1_ChIPseq_1; Homo sapiens; ChIP-Seq
library_construction_protocol: ~ 50 mg of blood perfused liver tissue were dissected and snap frozen in liquid nitrogen. Frozen liver tissues were minced with razor blades and homogenized by pushing through 18G needle followed by 21G needle to release nuclei. Nuclei were immediately crosslinked with 1% formaldehyde for 10 min at room temperature and then quenched with glycine. After two washes, the nuclei were lysed in nuclear lysis buffer (50 mM Tris-HCl pH 8.0, 1% SDS, 10 mM EDTA) containing 1x EDTA-free protease inhibitor cocktail (Roche) and kept on ice for 5 min. Immediately, the lysates were sonicated 17 times for 30 sec by using Bioruptor (Diagenode). After centrifugation at 17,000 rpm for 15 minutes at 4°C, 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 incubated with antibody overnight at 4°C. 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). 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 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, DNA was purified with QIAquick PCR Purification columns.   ChIP-seq experiments with human cell lines were similar. The differences are the cells were crosslinked in culture dishes with 1% formaldehyde, and then scraped off for cell lysis and chromatin fragmentation. 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 HiSeq to generate more than 20M reads per sample.