Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
SMAD4

Cell type

Cell type Class
Kidney
Cell type
HGrC1
NA
NA

Attributes by original data submitter

Sample

source_name
HGrC1 (SMAD2/3 KO)
cell line
HGrC1
chip antibody
Rabbit polyclonal anti-SMAD4 (Proteintech, 10231-1-AP)
genotype/variation
HGrC1 (knockout for SMAD2 and SMAD3)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Confluent cell plates of KGN or HGrC1 cells were washed in PBS and cells were released by treatment with trypsin. Cells were collected by centrifugation (300g, 5 minutes), washed with PBS and centrifuged again. Cells were cross-linked with 0.2-mM DSG for 30 min. at room-temperature followed by cross-linking with 1% formaldehyde for 5 min at room temperature. Glycine was added at a final concentration of 125mM to quench the formaldehyde. Cells was washed twice with ice cold PBS and harvested in SDS buffer (50mM TrisHCl pH 8.1, 100mM NaCl, 5mM EDTA pH 8.0, 0,2% NaN3, 0.5% SDS). Cells were spun down, resuspended in Triton X-IP buffer (100mM Tris pH 8.6, 0.3% SDS, 1.7% Triton X-100 and 5mM EDTA) and the chromatin was sonicated to get DNA fragments of <600bp (with average DNA fragment size of 300bp) and then cleared by centrifugation. 1mg (transcription factors) or 100 g of chromatin (histone modifications) was precleared with Protein G coupled Sepharose beads (GE Healthcare) for 3hrs and incubated with 0,5-10 g antibody overnight at 4 C. The next day, Protein G coupled sepharose beads were added and samples were incubated end-over-end for 4hrs at 4 C. Beads were washed for a total of five times in low salt buffer (20mM Tris-HCl pH 8.0, 1% Triton X-100, 0.1% SDS, 150mM NaCl, 2mM EDTA pH 8.0), two times in RIPA buffer (50mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP40, 0.1% SDS, 0.5% DOC, 1mM EDTA), and one time with high salt buffer (20mM Tris-HCl pH 8.0, 1% Triton X-100, 0.1% SDS, 500mM NaCl, 2mM EDTA pH 8.0). Beads were incubated with elution buffer (1% SDS, 0.1M sodium bicarbonate) at 65 C overnight to de-crosslink. The DNA was isolated using QIAquick PCR Purification kit (Qiagen) and eluted in 50 l EB buffer.) ChIP sequencing libraries were prepared for sequencing using standard Illumina protocols RNA and ChIP sequencing libraries were prepared for sequencing using standard Illumina protocols

Sequencing Platform

instrument_model
NextSeq 500

hg19

Number of total reads
66902264
Reads aligned (%)
82.7
Duplicates removed (%)
13.0
Number of peaks
1989 (qval < 1E-05)

hg38

Number of total reads
66902264
Reads aligned (%)
84.9
Duplicates removed (%)
11.3
Number of peaks
2371 (qval < 1E-05)

Base call quality data from DBCLS SRA