GSM5914632: Input_F1; Mus musculus; ChIP-Seq
Primary hepatocytes isolated rapidly from 10-week-old C57BL/6 male mice that had been deprived of food for 24 h with or without refeeding for 2 h were fixed with 1% formaldehyde in PBS for 10 min. After neutralization with 1 M glycine, the cells were suspended in ChIP buffer (5  mM HEPES-KOH [pH 8.0], 200  mM KCl, 1  mM CaCl2, 1.5  mM MgCl2, 5% sucrose, 0.5% Nonidet P-40, aprotinin [10  μg/ml], leupeptin [10  μg/ml], 1  mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail [cOmplete, Roche]), subjected to ultrasonic treatment with a Bioruptor (Diagenode), and digested with micrococcal nuclease (New England Biolabs) for 1 h at 37°C. After the addition of EDTA to a final concentration of 20  mM followed by an equal amount of RIPA buffer (50 mM Tris-HCl [pH 8.0], 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, 150 mM NaCl), the digested samples were centrifuged at 15,000 × g for 15  min at 4°C and the resulting supernatants were incubated overnight with rotation at 4°C with Dynabeads M-280 Sheep Anti-Rabbit IgG (Invitrogen) that had been conjugated with antibodies to FOXK1. After washing of the beads twice both with ChIP buffer and with Tris-EDTA buffer, bound proteins were eluted and cross-links were reversed by incubation for 24 h at 65°C with 1% SDS in Tris-EDTA buffer. DNA was purified with the use of a Qiaquick PCR Purification Kit (Qiagen) and was subjected to library preparation. ChIP-seq libraries were prepared with a SMARTer ThruPLEX DNA-seq Kit (Clontech) and NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England BioLabs).