Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
MITF

Cell type

Cell type Class
Epidermis
Cell type
A-375
Primary Tissue
Skin
Tissue Diagnosis
Melanoma

Attributes by original data submitter

Sample

source_name
Cultured A375 melanoma cells
cell line
A375 cells expressing empty vector
antibody
anti-MITF (Atlas Antibodies cat no. HPA003259)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Empty vector and PRL3 stable overexpressing cells were cultured to 80% confluency and harvested by dissociation with trypsin in PBS/EDTA. Cells were resuspended in PBS and immediately fixed in 1% formaldehyde in PBS for 10 minutes at room temperature. The fixation was neutralised by the addition of glycine and incubated for an additional 5 minutes. The cells were washed in cold PBS. Cell pellets were resuspended in 150 µl cold lysis buffer (1% SDS, 10mM EDTA, 50 mM Tris-HCl pH8.1, 1x protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitors (Roche) and fresh 1mM DTT) and supplemented with 1350 µl 1% Triton X IP dilution buffer (1% Triton X, 20mM Tris-HCl pH8.1, 150mM NaCl, 2mM EDTA, 1x protease inhibitor cocktail (Roche), 1x PhosSTOP phosphatase inhibitors (Roche) and fresh 1mM DTT and 1mM PMSF) and incubated on ice for 10 min. Lysed cells were sonicated on ice for 5x30 seconds with a probe sonicator followed by 50-60 cycles (30 sec on / 30 sec off) in a chilled bioruptor (Diagenode) to yield chromatin fragments ranging between 200 – 800 bp in length. For MITF ChIP, lysed cells were sonicated in a chilled SoniPrep150 probe sonicator for 15x30 seconds with a 30 seconds gap between burst. Sheared chromatin was centrifuged at max speed for 10 minutes at 4°C and the soluble supernatant transferred to new tubes. 500 ml chromatin samples were supplemented with 5 ml (5mg/ml) BSA. 10% of the input was stored and the rest was used for the immunoprecipitation. Anti-DDX21 (Proteintech; cat no. 10528-1-AP), RNA-POLII tot, RNA-POLII PS2, PS5 (Novus Biologicals) and MITF (Atlas Antibodies) were pre-bound to proteinG dynabeads (ThermoFisher) in 10% w/v BSA in PBS according to the manufacturer's instructions (Life Technologies) for approximately 1 h at 4°C with rotation following which free antibody was removed with 3 washes of cold 10% w/v BSA in PBS. Chromatin and proteinG beads were combined and incubated over night at 4°C (at a ratio of 500 ng of bead bound antibody per 1M cell equivalents of chromatin). Samples were washes at 4°C with rortation through the following series: 2 times in 1% Triton X IP dilution buffer, 2 times with ChIP wash A (50mM Hepes ph7.9, 500mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, 0.1% SDS. 1x protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitors (Roche) and fresh 1mM DTT) and 2 times with ChIP wash B (20mM Tris pH 8.0, 1mM EDTA, 250mM LiCl, 1% NP-40, 0.1% Na-deoxycholate, 1x protease inhibitor cocktail, 1x PhosSTOP phosphatase inhibitors (Roche) and fresh 1mM DTT). Finally, the samples were washed with TE (1mM EDTA ,10mM Tris pH8.0). The samples were resuspended in TE and supplemented with preheated 37°C Extraction buffer (0.1M NaHCO3 and 1%SDS), vortexed and incubated for 15 minutes at 37°C on a vibrating platform. The pH of the extracted chromatin was adjusted by adding 6ml 2M Tris-HCl pH6.8 following which both the ChIP and input samples were incubated with 20mg RNAseA at 65°C for 1 hour. Cross-links were reversed and the protein degraded by the addition of 20mg Proteinase K and incubation at 65°C for 6-8 h. Following removal of the dynabeads from the ChIP samples, DNA was purified using a Qiagen PCR cleanup kit following manufacturer's instructions. DNA libraries were prepared using the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® and NEBNext® Multiplex Oligos for Illumina® NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1) following manufacturer's instructions.

Sequencing Platform

instrument_model
NextSeq 550

hg19

Number of total reads
69396135
Reads aligned (%)
91.4
Duplicates removed (%)
6.1
Number of peaks
2423 (qval < 1E-05)

hg38

Number of total reads
69396135
Reads aligned (%)
93.7
Duplicates removed (%)
4.5
Number of peaks
3085 (qval < 1E-05)

Base call quality data from DBCLS SRA