GSM7846908: ChIP-Seq IGG_CLP; Mus musculus; ChIP-Seq
ChIP was performed on 200-400 mg of snapfrozen liver tissue. First, DynabeadsTM protein G (Thermo Fisher Scientific) were washed three times with BSA/PBS and coupled to a primary antibody against HNF4α (3 µg/IP sample; PP-H1415-00, R&D systems) or mouse IgG (3 µg/IP sample; sc-2025, SantaCruz), as an internal control, overnight at 4°C. Afterwards, the beads with coupled antibody were washed with BSA/PBS twice. Livers were homogenized and fixed in Farnham Lysis buffer with 1% formaldehyde, supplemented with protease inhibitor cocktail (Roche), for 10 min at RT. Released nuclei were isolated by centrifugation at 2000 rpm for 5 min and washed with PBS, supplemented with protease inhibitor cocktail (Roche). The nuclei were further lysed in RIPA buffer, supplemented with protease inhibitor cocktail (Roche), and sonicated (Bioruptor, Diagenode) at 4°C for 40 cycles (30s ON, 30s OFF) with high settings to yield 200-800 bp DNA fragments. Cell debris was removed by centrifugation at full speed for 20-30 min at 4°C. Sonication efficiency was checked (after reverse crosslinking with proteinase K for 1h at 65°C and DNA purification) on a 1.5% agarose gel. For immunoprecipitation (IP), antibody-coupled magnetic beads were added to the sonicated samples and incubated overnight at 4°C. Input samples containing sonicated lysate were used as a control during analysis. The day after, bead-antibody-DNA complexes were washed 5 times with LiCl wash buffer (100 mM Tris-HCl; pH 7.5, 500 mM LiCl, 1% IGEPAL, 1% sodium deoxycholate) at 4°C and 2 times with TE buffer (10 mM Tris-HCl; pH 7.5, 0.1 mM EDTA) at 4°C. Chromatin was eluted by incubating the IP samples with elution buffer (1% SDS, 0.1M NaHCO3) 1h at 65°C. Both IP and input samples were incubated with proteinase K for 4h at 65°C for reverse crosslinking and DNA was purified in 50 µL elution buffer according to the manufacturer's protocol by using the Monarch PCR&DNA Cleanup kit (Biolabs). DNA concentration was measured with Qubit Fluorometer using the Qubit 1x dsDNA high sensitivity kit (Thermo Fisher Scientific) 15 ng of purified chromatin was used to generate a ChIP-Seq library according to the manufacturer's protocol using NEBNext Ultra II DNA Library Prep kit (E7645L, NEB).