Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
SMAD3

Cell type

Cell type Class
Epidermis
Cell type
BJ
Primary Tissue
Skin
Tissue Diagnosis
Normal

Attributes by original data submitter

Sample

source_name
BJ Fibroblasts
cell line
BJ Fibroblasts
chip antibody
Anti-Smad3 antibody - ChIP Grade (ab28379)- Lot #- GR155884-8
treatment
BJ fibroblasts 5 days post induced viral expression of OSKM
genotype
WT

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
10 million cells were cross-linked with 10 mM DTBP (Sigma Aldrich) and 1% formaldehyde for 10 min at room temperature, with the formaldehyde then quenched by the addition of glycine (125mM final concentration) and the cells were then washed twice with ice-cold PBS. Cell lysis was carried out with a buffer containing 10 mM Tris-Cl (pH 8), 100 mM NaCl, 10 mM EDTA, 0.25% Triton X-100 and protease inhibitor cocktail (Roche). Cells were then resuspended in 1% SDS lysis buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 1% SDS, 2 mM EDTA, 1% Triton X-100, 0.1% NaDOC and protease inhibitor cocktail. The suspension was nutated for 15 mins at 4°C before spinning down to collect the chromatin pellet. The pellet was then washed twice with 0.1% SDS lysis buffer containing 50 mM HEPES-KOH (pH 7.5), 150 mM NaCl, 0.1% SDS, 2 mM EDTA, 1% Triton X-100, 0.1% NaDOC, 1 mM PMSF and protease inhibitor cocktail. Sonication was conducted using the Bioruptor (20 cycles, 30s pulses on with 90s pulses off). The chromatin solution was clarified by centrifugation at 20,000 g at 4°C for 45 mins and then pre-cleared with Dynabeads protein G (Life Technologies) for 2 hrs. The pre-cleared chromatin sample was incubated with 50 μl of Dynabeads protein A loaded with 10 μg antibody overnight. The beads were then washed three times with 0.1% SDS lysis buffer, once with 0.1% SDS lysis buffer/0.35 M NaCl, once with 10 mM Tris-Cl (pH 8), 1 mM EDTA, 0.5% NP40, 0.25 LiCl, 0.5% NaDOC, and once with TE buffer (PH8.0). The immunoprecipitated material was eluted from the beads by heating for 45 min at 68°C in 50 mM Tris-Cl (pH 7.5), 10 mM EDTA, 1% SDS. To reverse the crosslinks, samples were incubated with 1.5 μg/ml Pronase at 42°C for 2 hr followed by 67°C for 6 hr. The samples were then extracted with miniElute DNA purification kit (Qiagen).

Sequencing Platform

instrument_model
Illumina HiSeq 2000

hg19

Number of total reads
9144205
Reads aligned (%)
82.0
Duplicates removed (%)
16.3
Number of peaks
228 (qval < 1E-05)

hg38

Number of total reads
9144205
Reads aligned (%)
83.7
Duplicates removed (%)
15.9
Number of peaks
264 (qval < 1E-05)

Base call quality data from DBCLS SRA