Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
SMAD2

Cell type

Cell type Class
Cardiovascular
Cell type
Aortic smooth muscle cells
NA
NA

Attributes by original data submitter

Sample

source_name
SMAD_ChIPSeq
cell type
aortic smooth muscle cells
treatment
TGF beta treatment
chip antibody
SMAD2

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
10x10^6 cells were first cross-linked with 1.5 mM EGS in PBS for 30 mins, followed by 1% paraformaldehyde for 10 minutes at room temperature. Glycine was added at a final concentration of 125 mM for 5 minutes at room temperature in order to quench the crosslinking reaction. The cross-linked material was washed once each with Buffer 1 (0.25% Triton, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH7.5) and Buffer 2 (200 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, 10 mM Tris pH7.5) before resuspension in Shearing Buffer (0.1% SDS, 1 mM EDTA, 1 mM Tris-HCl pH7.6) with 1X Complete Protease Inhibitor Cocktail (Roche). Cell pellet was lysed in 130 μl SDS lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA, 1% SDS, 50 mM PMSF) and sonicated using an S220 Focused-ultrasonicator (Covaris) to generate 200 to 600 bp fragments. Fragmented chromatin was centrifuged at 10,000 g for 5 minutes and the supernatants were diluted in ChIP dilution buffer (1% Triton, 150 mM NaCl, 20 mM Tris pH 8.0). Immunoprecipition was performed by rotating samples at 4°C with magnetic beads (Dynabeads Protein A or G, Invitrogen) pre-bound with 5 μg of antibody. The beads were washed once each with low salt buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.1% SDS, 0.5% Deoxycholate, 1% NP40, 1 mM EDTA), high salt buffer (50 mM Tris pH 8.0, 500 mM NaCl, 0.1% SDS, 0.5% Deoxycholate, 1% NP40, 1 mM EDTA), LiCl wash buffer (50 mM Tris pH 8.0, 250 mM LiCl, 0.5% Deoxycholate, 1% NP40, 1 mM EDTA), Morohashi RIPA buffer (50 mM Tris pH7.5, 150 mM NaCl, 5 mM EDTA, 0.5% NP40, 0.1% SDS), DOC/Triton Buffer (25 mM Tris pH7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton-X-100, 0.5% DOC), and twice with Tris-EDTA buffer (10 mM Tris pH 8.0, 1 mM EDTA). All washes took place on a rotator for 10 minutes at 4°C. Beads were treated twice with 100μl elution buffer for 15 minutes on a shaker at room temperature, to obtain a total of 200μl eluate. 8 μl of 5M NaCl was added to the eluate and the sample was reverse cross-linked overnight at 55°C. DNA was then purified using the QIAquick PCR Purification Kit (Qiagen). ChIP DNA samples was used in a 23.3 μl combined end repair and A-tailing reaction using a KAPA Hyper Prep Kit (Kapa Biosciences) for 30 minutes at 20°C followed by 30 minutes at 65°C. 10 μl of ligase buffer, 3.7 μl of Adapters and 3.3 μl ligase (KAPA Hyper Prep Kit) were added and incubated at 20°C for 4 hours. Double-stranded DNA fragments were purified from this reaction using KAPA Pure Beads (Kapa Biosciences) and eluted in 22 μl 10 mM Tris pH8.0. Libraries were generated by PCR and size-selected on a 2% E-Gel EX agarose gel (Invitrogen) and fragments between 150 and 400 bp were extracted using a QIAEX II Gel Extraction Kit (Qiagen) performed at room temperature.

Sequencing Platform

instrument_model
Illumina HiSeq 4000

hg19

Number of total reads
14914365
Reads aligned (%)
112.9
Duplicates removed (%)
25.2
Number of peaks
337 (qval < 1E-05)

hg38

Number of total reads
14914365
Reads aligned (%)
115.3
Duplicates removed (%)
24.8
Number of peaks
600 (qval < 1E-05)

Base call quality data from DBCLS SRA