GSM4217713: 4C-HA-MITF input_2; Homo sapiens; ChIP-Seq
Hermes cells were transduced by lentiparticles carrying pLX3xHAvar4mCherry. Stable cell lines expressing the 3xHA tagged MITFvar4 were subsequently sorted based on the presence of mCherry and used further. Chromatin immuno-precipitation sequencing (ChIPseq) experiments were performed twice. Cross-linking was performed with 1% formaldehyde for 8min. Cross-linking was stopped with 125mM glycine for 5min. Cells were rinsed twice in phosphate buffered saline (PBS), harvested using trypsination and sedimented. Cells were resuspended in ice-cold lysis buffer (1% SDS, 10mM EDTA, 50mM Tris–HCl, pH 8.0) containing protease and phosphatase inhibitors. Cells were sonicated at 4°C using a Covaris S2 instrument to yield chromatin fragments of 300–500 bp. Chromatin was cleared by centrifugation, concentration determined by A260, and chromatin was diluted in RIPA buffer (0.1% SDS, 0.1% Na-deoxycholate, 1% Triton X-100, 1mM EDTA, 0.5mM EGTA, 140mM NaCl, 10mM Tris–HCl, pH 8.0) to 2 A260 units. Each immunoprecipitation was performed in 250μl RIPA buffer with 1μg antibody overnight at 4°C. The antibody: anti-HA-tag (12CA5, Roche) was used. Antibody-bound chromatin was precipitated using Dynabeads protein G (Invitrogen) and washed 3 times in RIPA buffer, once in TE buffer (10mM Tris–HCl, pH 8.0, 10mM EDTA) and eluted in 1% SDS with 100mM NaHCO3. Eluted chromatin was incubated at 65°C with proteinase K (Sigma-Aldrich; 50μg/ml) over night, and DNA was purified by phenol-chloroform-isoamylalcohol extraction (25:24:1) and once with chloroform/isoamylalcohol (24:1) followed by ethanol precipitation. DNA was dissolved in H2O and used for high-throughput sequencing using an Illumina Hiseq 2500 sequencer. Libraries were prepared according Illumina's instructions accompanying the TruSeq library preparation kit.
GEO Accession
GSM4217713