Chromatin Immunoprecipitation was performed as previously described (Jimeno-González et al., 2015). Briefly, serum-starved RPE-1 cells were crosslinked with 1% formaldehyde for 10 minutes at 37°C. Crosslinking reaction was quenched with 125mM glycine for 5 minutes. Cell pellets were resuspended in 2.5 mL lysis buffer A (5 mM Pipes pH 8.0, 85 mM KCl, 0.5% NP40) supplemented with protease inhibitors and incubated for 10 minutes on ice. Chromatin was obtained by centrifugation at 4000 rpm for 5 minutes at 4°C. Nuclear fraction was resuspended in 1 mL of lysis buffer B (50 mM Tris HCl pH 8.1, 1% SDS, 10 mM EDTA, 1 mM PMSF) supplemented with protease inhibitors. Chromatin was sonicated using a Bioruptor (Diagenode, UCD-200), 10 cycles of 30” sonication (high level) and 30” of pause on ice-cold water. 50 μL of sonicated chromatin was reverse-crosslinked using Proteinase K in PK buffer (0.5%SDS, 50mM Tris-Cl, 100mM NaCl, 1mM EDTA) at 65°C overnight. After phenol chloroform extraction, DNA fragmentation was analyzed on 1.2% agarose gel. For each inmunoprecipitation, 20 μg of chromatin and 4 μg of the specific antibody was used in IP buffer (0,1% SDS, 1% TX-100, 2mM EDTA, 20 mM TrisHCl pH8, 150 mM NaCl) at 4°C o/n and then with 40 μL of pre-blocked (1 mg/ml BSA) Dynabeads protein A (ThermoFisher). Beads were sequentially washed with Wash buffer 1 (20 mM Tris HCl pH 8.1, 0.1% SDS, 1% Triton x-100, 2 mM EDTA, 150 mM NaC), Wash buffer 2 (20 mM Tris HCl pH 8.1, 0.1% SDS, 1% Triton x-100, 2 mM EDTA, 500 mM NaCl) Wash buffer 3 (10 mM Tris HCl pH 8.1, 1% NP-40, 1% NaDoc, 1 mM EDTA, 250 mM LiCl), and twice with TE-buffer (10 mM Tris-HCL pH8, 1 mM EDTA pH8). ChIPmentation was carried out as previously described (Schmidl et al., 2015), using Tn5A Enzyme provided by the Proteomic Service of CABD (Centro Andaluz de Biología del Desarrollo). Tagmented DNA was then eluted with 1% SDS in TE at 65°C for 10 minutes and protein was degraded with Proteinase K for 2 hours at 37°C. DNA was purified using QIAGEN PCR purification Kit (QIAGEN, 28106). Libraries were amplified for N-1 cycles (being N the optimum Cq determined by qPCR reaction) using NEBNext High-Fidelity Polymerase (New England Biolabs, M0541). Libraries were purified with Sera-Mag Select Beads (GE Healthcare, 29343052) and sequenced using Illumina NextSeq 500 and single-end configuration.

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