GSM8119229: Samp4, Mouse Hepatocytes, H4K16ac, BA, ChIP  [MBA4H4K16ac]; Mus musculus; ChIP-Seq
Primary mouse hepatocytes were isolated using the Liver Perfusion Kit mouse & rat (Miltenyi Biotec, 130-128-030) following the manufacturer's instruction. The lobus hepatis dexter was dissected and used for further perfusion procedure using the pre-programmed protocol 37C_m_LIPK_1 via the gentleMACS Octo Disocciator system with heaters (MACS Technology, Miltenyi Biotec). The perfused liver lobe was homogenized via the gentleMACS program LIPK_HR_1. Enzymatic digestion was stopped by adding 5 ml attachment medium (Williams E media (Life Technologies, 22551089) supplemented with 2.24 g/l NaHCO3, 100 U/100 µM penicillin/streptomycin, 100 µM dexamethasone (Biozol, FBM-10-1134), 2 mM L- glutamine and 10% FCS). The cell suspension was filtered through a 70 µm cell strainer. The gentleMACS C tube and the cell strainer were subsequently rinsed twice with 4 ml of attachment medium. Cells were collected via centrifugation (5 min, 77 xg, 4°C). The supernatant was discarded and the pellet was washed twice with ice-cold PBS. 106 cells were seeded into each well of a 6 well plate in attachment medium. After 3 hours, medium was changed and cells were cultured  in 5% CO2 at 37°C using Williams E media supplemented with 2.24 g/l NaHCO3, 100 U/100  µM penicillin/streptomycin, 100 µM dexamethasone and 2 mM L-glutamine. We performed the restriction enzyme-based labelling of chromatin (RELACS), coupled to  deep sequencing. RELACS is a variant of the standard chromatin immunoprecipitation (ChIP)  protocol. For each sample, 1 x 106 cells were fixed in 1% formaldehyde for 14 minutes and  quenched with the addition of 125 mM glycine for 1 minute. Cells were washed three times  with PBS supplemented with cOpmlete protease inhibitor cocktail (Roche, 11836145001) and  10 mM sodium butyrate (Sigma Aldrich, 303410), followed by harvesting via scraping from  plates. ChIP was performed as previously described42, except that no barcode ligation and  pooling was performed. Nucleus isolation was performed for 4.5 minutes at peak power 75  Watt, duty factor 2% and 200 cycles/burst using the Covaris E220 instrument.  Immunoprecipitation (IP) was performed using 2 µg of antibody and protein A/G Dynabeads  (Thermo Scientific, 88802). For each IP, an equal amount of chromatin was used per sample.  Chromatin quantification was performed by Nanodrop (Thermo Fisher Scientific). The  immunoprecipitated DNA was quantified by Qubit 4 Fluorometer (Thermo Scientific) prior to  library preparation. Sequencing libraries were prepared using the NEBNext Ultra library  preparation kit (New England BioLab, E7645S), and then assessed for quality and quantity via  the BioAnalyzer (Agilent). Generated libraries were sequenced on an Illumina NovaSeq  instrument (100 bp, paired-end). Chromatin Immunoprecipitating Sequencing (ChIP-Seq) from Primary Mouse Hepatocytes