Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Histone
Antigen
H3K4me3

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
M381
cell line
Patient-derived melanoma cell line M381
treatment
Untreated M381 cells
chip antibody
H3K4me3 (Millipore, 07-473)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Briefly, cells were cultured to ~80% confluency in a petri dish containing 10 mL of growth media, and then fixed in 1% formaldehyde by adding 275 μl of 37% formaldehyde for 10 minutes to cross-link protein–DNA complexes at room temperature. The unreacted formaldehyde was quenched by adding Glycine to a final concentration 0.125 M. Gently swirl dish to mix. The nuclear pellet was isolated with Cell Lysis Buffer. The pellet was resuspended with 500 μl SDS Lysis Buffer containing 1X Protease Inhibitor Cocktail II before sonication for 4 min (10 s on, 30 s off, 10% strength in a Bioruptor to yield DNA fragments of 0.2-1.0 kb in length. The lysates were cleared by centrifugation (12,000g for 10 min at 4 °C) and diluted tenfold in ChIP dilution buffer to decrease the concentration of SDS. After keeping 10% of the sample as input, 500 μl supernatant was incubated overnight at 4 °C with antibody and 20 μL of fully resuspended protein A/G magnetic beads. The washing, elution, reverse cross-linking, and purification steps were performed according to manufacturer's description. ChIPed Eluted DNA was quantified by Qubit dsDNA HS Assay Kit. As for ATAC-seq, the previous published protocol (Buenrostro et al., 2015) were used for cell lysis, tagmentation and DNA purification. For RNA-seq, total RNA was extracted from cell pellets using RNeasy Mini Kit (Qiagen). ChIP-seq libraries were prepared with Kapa DNA HyperPrep Kit (Kapa, Cat KK 8700) according to the manufacturer's protocol. Briefly, 5-10 ng of immunoprecipitated DNA was underwent end-repaired, A tailing and adaptor ligation. A 10 cycles of PCR was performed to produce the final sequencing library. As for ATAC, the Tn5 treated DNA was amplified with a 5-cycle PCR in 50µl reaction volume. The tubes were removed from thermocycler and used 5 µl of partially amplified library to perform qPCR to determine how many additional PCR cycles were needed. For the samples in this study, an additional 4-5 cycles of PCR was performed on the remaining 45ul of each partially amplified product. 1.8X AmpurXP beads purification was used for the final PCR cleanup. The libraries were validated with the Agilent Bioanalyzer DNA High Sensitivity Kit, and quantified with qPCR. For RNA-seq, sequencing libraries were prepared with TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, USA) with minor modifications.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

hg38

Number of total reads
46890412
Reads aligned (%)
76.9
Duplicates removed (%)
10.9
Number of peaks
2285 (qval < 1E-05)

hg19

Number of total reads
46890412
Reads aligned (%)
74.7
Duplicates removed (%)
14.9
Number of peaks
2015 (qval < 1E-05)

Base call quality data from DBCLS SRA