Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K4me1

Cell type

Cell type Class
Epidermis
Cell type
Melanoma
MeSH Description
A malignant neoplasm derived from cells that are capable of forming melanin, which may occur in the skin of any part of the body, in the eye, or, rarely, in the mucous membranes of the genitalia, anus, oral cavity, or other sites. It occurs mostly in adults and may originate de novo or from a pigmented nevus or malignant lentigo. Melanomas frequently metastasize widely, and the regional lymph nodes, liver, lungs, and brain are likely to be involved. The incidence of malignant skin melanomas is rising rapidly in all parts of the world. (Stedman, 25th ed; from Rook et al., Textbook of Dermatology, 4th ed, p2445)

Attributes by original data submitter

Sample

source_name
Melanoma Tumor Sample
cell type
Tissue
chip-antibody
H3K4me1 Abcam ab8895

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP assays were performed as described previously in Terranova et al (An Integrated Platform for Genome-wide Mapping of Chromatin States Using High-throughput ChIP-sequencing in Tumor Tissues; JoVE 2018). Briefly, 50mg of tissue or 20million cells were harvested via cross-linking with 1% (wt/ vol) formaldehyde for 10 min at 37 °C with shaking. After quenching with 150 mM glycine for 10 min at 37 °C with shaking, cells were washed twice with ice-cold PBS and frozen at −80 °C for further processing. Cross-linked pellets were thawed and lysed on ice for 30 min in ChIP harvest buffer (12 mM Tris-Cl, 1 × PBS, 6 mM EDTA, 0.5% SDS) with protease inhibitors (Sigma). Lysed cells were sonicated with a Bioruptor (Diagenode) to obtain chromatin fragments (~200–500 bp) and centrifuged at 15,000 × g for 15 min to obtain a soluble chromatin fraction. In parallel with cellular lysis and sonication, antibodies (5 μg/3 × 106 cells) were coupled with 30 μl of magnetic protein G beads in binding/blocking buffer (PBS + 0.1% Tween + 0.2% BSA) for 2 h at 4 °C with rotation. Soluble chromatin was diluted five times using ChIP dilution buffer (10 mM Tris-Cl, 140 mM NaCl, 0.1% DOC, 1% Triton X, 1 mM EDTA) with protease inhibitors and added to the antibody-coupled beads with rotation at 4 °C overnight. After washing, samples were treated with elution buffer (10 mM Tris-Cl, pH 8.0, 5 mM EDTA, 300 mM NaCl, 0.5% SDS), RNase A, and Proteinase K, and cross-links were reversed overnight at 37. Immune complexes were then washed five times with cold RIPA buffer (10mM Tris–HCl, pH 8.0, 1mM EDTA, pH 8.0, 140mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% DOC), twice with cold high-salt RIPA buffer (10mM Tris–HCl, pH 8.0, 1mM EDTA, pH 8.0, 500mM NaCl, 1% Triton X-100, 0.1% SDS, 0.1% DOC), and twice with cold LiCl buffer (10mM Tris–HCl, pH 8.0, 1mM EDTA, pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% DOC). ChIP DNA was purified using AMPure XP beads (Agencourt) and quantified using the Qubit 2000 (Invitrogen) and Bioanalyzer 1000 (Agilent). Libraries for Illumina sequencing were generated following the New England BioLabs (NEB) Next Ultra DNA Library Prep Kit protocol. A total of 10 cycles were used during PCR amplification for the generation of all ChIP-seq libraries. Amplified ChIP DNA was purified using double-sided AMPure XP to retain fragments (~200–500 bp) and quantified using the Qubit 2000 and Bioanalyzer 1000 before multiplexing. Libraries for Illumina sequencing were generated following the New England BioLabs NEBNext Ultra DNA Library Prep Kit protocol. A total of 10x cycles were used during PCR amplification for the generation of all ChIP-seq libraries. Amplified ChIP DNA was purified using double-sided AMPure XP to retain fragments ~200 to 500bp and quantified using the Qubit 2000 and TapeStation 2000 before multiplexing.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg38

Number of total reads
55887890
Reads aligned (%)
97.9
Duplicates removed (%)
3.9
Number of peaks
153 (qval < 1E-05)

hg19

Number of total reads
55887890
Reads aligned (%)
97.6
Duplicates removed (%)
4.2
Number of peaks
574 (qval < 1E-05)

Base call quality data from DBCLS SRA