GSM2055890: H1 ChIP-seq in FoxAflox;Alfp-Cre Mice; Mus musculus; ChIP-Seq
The nuclear suspension was layered onto a 1.5 ml cushion of 1:1 of buffer A: buffer B (15 mM Hepes [pH 7.6], 60 mM KCl, 15 mM NaCl, 0.1 mM EDTA, 0.1 mM EGTA, 2.1 M sucrose, 0.15 mM 2-mercaptoethanol) in 15 ml Corex tubes, centrifuged at 7.5-10K rpm for 10 min at 4°C, and the supernatant was removed.  The pellets were suspended in 5-10 ml Buffer A and layered again onto 1:1 of buffer A:B, and centrifuged at 5K rpm for 10 min at 4°C, and the supernatant was removed.  The pellet was suspended into 4 ml sonication buffer (50 mM Tris [pH8], 2 mM EDTA, 0.5 % N-Lauroylsarcosine, complete EDTA-free protease inhibitor cocktail (Roche), 0.5 mM PMSF) for Bioruptor sonication, or about 8 ml sonication buffer as adjusted the concentration at 2-2.4 x 107 nuclei/ml for Covaris sonication, and incubated for 5 min at room temperature, then 5 min on ice. The nuclear lysate was transferred to milliTUBE ATA Fiber tubes (#520130) and sonicated for 8 min (Temperature: 5-9 C, PP: 200, DF 10, CB 200) using Covaris S220. A portion of chromatin was reverse-crosslinked and confirmed, by agarose gel electrophoresis, that their average DNA fragment size was around 200 bp.  RNase A was added to 12.5-25 ng/ul and incubated at room temperature for 10 min.  Debris was removed by centrifugation at 14K rpm for 10 min at 4°C.  The supernatant was dialyzed in TE (10 mM Tris [pH8], 1 mM EDTA) using 3500 MWCO Slide-A Lyzer Dialysis cassette (Thermo Scientific) at 4°C for several hours, and move into new TE and dialyzed overnight. 20 ul (for H1 ChIP) or 50 ul (for FoxA3, C/EBPβ, and HNF4α ChIP) Dynabeads Protein G (Life technologies) were washed three times with 500 ul blocking solution (0.5 % w/v BSA in PBS).  The beads were resuspended in 20 ul or 50 ul blocking solution and saturated with 2.5 ug H1 antibody (Millipore, 05-457), 5 ug FoxA3 antibody (Santa Cruz, sc-5361), 5 ug C/EBPβ antibody (Santa Cruz, sc-150), 5 ug HNF4α antibody (Abcam, ab42898) by rotating the mixture for 2-6 hr at 4°C.  The antibody-beads were washed two times with 500 ul blocking solution and two times with 500 ul ChIP buffer (50 mM Hepes-KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 0.1 % Triton X-100, 1 mM PMSF, Complete EDTA free protease inhibitor cocktail (Roche)).  ChIP was performed in 100 ul (for H1) or 250 ul (for FoxA3, C/EBPβ, and HNF4α) ChIP buffer containing antibody-beads mixture and 4-12.5 ug (for H1) or 50 ug (for FoxA3, C/EBPβ, and HNF4α) of sonicated chromatin, which was based on O.D. A260 of reverse-crosslinking DNA, and rotated for 16 hr at 4°C.  The ChIP-beads mixture was washed for 5 min with rotation successively two times with 500 ul ChIP buffer, one time with 500 ul ChIP buffer plus 500 mM NaCl, two times with 500 ul LiCl solution (10 mM Tris-HCl [pH 8], 250 mM LiCl, 1 mM EDTA, 0.5% NP-40), and two times with 500 ul TE plus 0.1% NP-40.  The chromatin was eluted from the beads in 67 ul TES (50 mM Tris-HCl [pH 8], 10 mM EDTA, 1% SDS) and incubated at 65°C for 15 min.  The supernatant was placed in a fresh tube and the elution was repeated two more times, combining all supernatants.  The eluted chromatin and input chromatin were reverse-crosslinked by adding NaCl (final 200 mM) and incubating at 65°C for 4 to 16 hr. The chromatin was treated with 0.2 mg/ml Proteinase K and incubated at 37°C for 2 hr.  DNA was purified by phenol-chloroform extraction followed by ethanol precipitation. We prepared multiplexed libraries from two biological replicates of H1, FoxA3, C/EBPβ and HNF4α ChIP and Input from FoxA1/FoxA2 flox;Alfp-Cre and WT.  We used NEBNext Ultra DNA Library Prep Kit for Illumina (#E7370). Libraries from FoxA1/FoxA2 flox;Alfp-Cre and WT were sequenced as 75-bp single-end, using Illumina NextSeq500.
GEO Accession
GSM2055890