title: GSM4217713: 4C-HA-MITF input_2; Homo sapiens; ChIP-Seq
library_construction_protocol: 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.
attributes.tag: GEO Accession
attributes.value: GSM4217713