GSM3526293: ChIP_rescue_R133C; Mus musculus; ChIP-Seq
Sub-confluent MEFs overexpressing FLAG-HA tagged MeCP2 were crosslinked with 0.4% formaldehyde for 10 min and quenched with 150 mM glycine for 10 min at room temperature. Cells were lysed in hypotonic buffer (10 mM Tris-HCl pH 7.65, 1.5 mM MgCl2, 10 mM KCl) and disrupted by dounce homogenizer. The cytosolic fraction was separated from the nuclei by centrifugation at 4°C. Nuclei were re-suspended in sucrose buffer (20 mM Tris-HCl at pH 7.65, 15 mM KCl 60 mM NaCl, 0.15 mM spermine, 0.5 mM spermidine) adjusted with high-salt buffer (20 mM Tris-HCl pH 7.65, 25 % glycerol, 1.5 mM MgCl2, 0.2 mM EDTA, 900 mM NaCl) to get a final NaCl concentration of 300 mM. The nuclear-soluble fraction was recovered by centrifugation at 4°C. The pellet containing chromatin fraction was incubated in sucrose buffer containing 1 mM CaCl2, and MNase (2.5 U/g of cells) 10 min at 37°C. Digestion was stopped with 4 mM EDTA and soluble chromatin was recovered by centrifugation at 4°C. Tagged MeCP2 was immunoprecipitated with anti-Flag M2-agarose (Sigma), eluted with Flag peptide (0.5 mg/mL), further affinity-purified with anti-HA antibody-conjugated agarose, and eluted with HA peptide (1 mg/mL). The HA and Flag peptides were first buffered with 50 mM Tris-Cl (pH 8.5), then diluted to 4 mg/mL in TGEN 150 buffer (20 mM Tris at pH 7.65, 150 mM NaCl, 3 mM MgCl2, 0.1 mM EDTA, 10% glycerol, 0.01% NP40), and stored at -20°C until use. Between each step, beads were washed in TGEN 150 buffer. MeCP2-bound chromatin was submitted to RNase and proteinase K digestion, and DNA was extracted by phenolchloroform. Libraries were prepared using the Diagenode MicroPlex Library Preparation kit v2, and sequenced on Illumina Hiseq 4000 sequencer as single-end 50 bp reads following Illumina's instructions.
GEO Accession
GSM3526293