GSM7286713: ChIp_H2AZ_2xMinikirikou_12-7; Gallus gallus; ChIP-Seq
We cross-linked 30 x 106 exponential growing cells by incubation for 5 minutes with 1% freshly prepared formaldehyde (Thermo Fisher Scientific, #28908) at room temperature. Fixation was stopped by adding 0.125 M glycine-PBS for five minutes at room temperature. After three ice cold PBS washing, nuclei were extracted in lysis buffer (10mM Tris-HCl, pH7.5, 10mM NaCl, 3mM MgCl2, 0.2% triton X-100, 0.5mM EGTA, 1mM DTT, 1x protease inhibitor cocktail (Sigma, #P8340)) for 5min on ice, 15 centrifuged and resuspended in digestion buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 1 mM CaCl2, 1x protease inhibitor cocktail (Sigma, #P8340)). Micrococcal Nuclease (MNase; Thermo Fisher Scientific, #EN0181) digestions were performed for 15 minutes at 37°C. The final concentration of 160 U/mL was used for ChIp coverage analyses. The reaction was stopped by adding 0.1 volume of stop buffer (200 mM EDTA pH 8.0, 40 mM EGTA pH 8.0). Chromatin was then sonicated during 30 sec at 7°C in a buffer adjusted to a final SDS concentration of 0.1% with a Covaris sonicator using 75W intensity, 200 cycles per burst, 5% of duty cycle. For nucleosome coverage analyses, DNA molecules were 25 recovered after RNAse A and proteinase K digestion and phenol-chloroform extraction and ethanol precipitation. Chromatin was then adjusted in 1x IP buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton-X100 and 0.1% SDS). Immunoprecipitation was performed overnight at 4°C, in a final volume of 300 μl of 1X IP buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA pH 8.0, 150 mM NaCl, 1% Triton-X100 and 0.1% SDS) on an amount of MNAse digested chromatin corresponding to 10 μg of DNA, with anti-H2A.Z antibody (Active Motif, #39013), according to manufacturer recommendations. Immuno-complexes were pulled down with 50 μl of protein-G coated magnetic beads (Thermo Fisher Scientific, Dynabeads protein G, #10004D) per sample. Beads and immuno-complexes were incubated for 2 h at 4°C, on a rotating wheel. The beads were then washed once with 1× IP buffer, twice with wash B buffer (20 mM Tris pH 8.0, 2mM EDTA pH 8.0, 500 mM NaCl, 0.25% Triton X-100) and then twice with 1× TE buffer pH 8.0. The DNA was eluted by incubating the beads for 2 h at 37°C with 250 μl 1× TE buffer pH 8.0, to which we added 1% SDS and 0.5 mg/ml proteinase K. Cross-linking was reversed by overnight incubation at 65°C, and samples were further treated with 10 μg of RNase A for 15 min at 37°C, and with 20 μg of proteinase K for 1 h at 56°C. DNA was purified by phenol–chloroform 5 extraction, precipitated in alcohol, resuspended in 100 μl TE and quantified by QuBit (dsDNA HS assay kit, Thermo Fisher Scientific, #Q32851). Input samples were obtained from MNAse digested chromatin after RNAse A and proteinase K digestion, phenol-chloroform extraction and ethanol precipitation. Sequencing libraries were prepared using NEBNext Ultra II DNA library prep Kit for Illumina (NEB #E7645S) according to the manufacturer's instructions for MNase, ChIp and input libraries. Samples were not subjected to size selection, but only cleaned-up for adaptor-ligated DNA using SPRISelect Reagent kit (Beckman coulter #B23317), to ensure a MNase digested small fragment overall recovery. Libraries were prepared with a starting DNA input of 10 ng. We used a 10 fold diluted adaptor for the adaptor ligation. The library amplification was performed using NEB-Next Multiplex Oligos for Illumina (NEB #E7710S) with different NEB-Next index primers and the NEB-Next Universal PCR Primer (NEB #E6861A) with eight PCR cycles.