Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
MITF

Cell type

Cell type Class
Epidermis
Cell type
Hermes 3C
NA
NA

Attributes by original data submitter

Sample

source_name
Hermes4c - lentiviral transduction of a HA-tagged MITF-M construct (pLX3xHAvar4mCherry)
cell line
Hermes4c
antibody
anti-HA-tag (12CA5, Roche)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
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.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg19

Number of total reads
53516699
Reads aligned (%)
69.8
Duplicates removed (%)
28.2
Number of peaks
72825 (qval < 1E-05)

hg38

Number of total reads
53516699
Reads aligned (%)
71.3
Duplicates removed (%)
27.0
Number of peaks
73061 (qval < 1E-05)

Base call quality data from DBCLS SRA