Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
RNF2

Cell type

Cell type Class
Epidermis
Cell type
Melanocytes
MeSH Description
Mammalian pigment cells that produce MELANINS, pigments found mainly in the EPIDERMIS, but also in the eyes and the hair, by a process called melanogenesis. Coloration can be altered by the number of melanocytes or the amount of pigment produced and stored in the organelles called MELANOSOMES. The large non-mammalian melanin-containing cells are called MELANOPHORES.

Attributes by original data submitter

Sample

source_name
Hmel human melanocytes, RNF2 ChIP
cell line
HMEL-BRAFV600E
cell type
immortalized melanocytes
chip antibody
V5 (Abcam ab9116, lot# 754608)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells (5 million per antibody) were crosslinked using 1% paraformaldehyde for 10mins at 37 degrees. Reaction was quenched by 0.125M glycine for 5mins, cells washed with PBS and stored at -80oC. Next day cells were thawed on ice and lysed with RIPA buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 140mM NaCl, 1% Triton x-100, 0.2%SDS, 0.1% DOC) for 10min on ice. Sonication was performed using Branson Sonifier 250 to achieve shear length of 200-500bp. Antibodies [Rabbit IgG, V5 (Abcam)] were bound to Dynabeads for 1 hr at 4oC. Extracts were then incubated overnight with antibody-Dynabead mixture. Immune complexes were then washed 5 times with RIPA buffer, once with RIPA-500 (RIPA with 500mM NaCl) and once with LiCl wash buffer (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% DOC). Elution and decrosslinking was performed in direct elution buffer (10mM Tris-Cl pH 8.0, 5mM EDTA, 300mM NaCl, 0.5% SDS) by incubating immune complexes at 65oC for 4-16hrs. Proteinase K (20mg/ml) and RNaseA treatment was performed and DNA cleaned up using SPRI beads (Beckman-Coulter). Libraries were prepared using NEB reagents as described (Garber et al. 2012). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3' end. After adapter ligation, DNA was PCR amplified with Illumina primers for 12 cycles and library fragments of ~300-500 bp (insert plus adaptor and PCR primer sequences) were size selected by SPRI beads. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the HiSeq 2000 following the manufacturer's protocols.

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
31043585
Reads aligned (%)
65.3
Duplicates removed (%)
5.4
Number of peaks
4506 (qval < 1E-05)

hg38

Number of total reads
31043585
Reads aligned (%)
66.1
Duplicates removed (%)
4.5
Number of peaks
4630 (qval < 1E-05)

Base call quality data from DBCLS SRA