 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 11, 2018 |
| Title |
TQ324-SMAD3 |
| Sample type |
SRA |
| |
|
| Source name |
HCASMC
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human coronary artery smooth muscle cells (HCASMC)
chip antibody: SMAD3
|
| Growth protocol |
HCASMC were grown in the presence of serum and additives.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
HCASMC were crosslinked in the dish, 2 million cells, and harvested.
ChIP was performed using the SMAD3 antibody Abcam, ab28379, and library was prepared using standard procedures. Briefly, DNA was prepared for end repair (Lucigen Endi-it, ER0720) and “A” tailing (NEB Klenow, M0212S), adaptor ligation (Promega, M180A), and library amplification (NEB, M0531S). ChIP-seq libraries were sequenced on HiSeq X10 for 150-bp paired-end sequencing.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Quality control of ChIP-seq data was performed using Fastqc, and then low-quality bases and adaptor contamination were trimmed by cutadapt. After quality control and data filtering, data was mapped to hg19 using BWA mem algorithm. Duplicate reads were marked by Picard Markduplicate module and removed with unmapped reads by samtools view -f 2 -F 1804. MACS2.1.1 was used for peaks calling with default parameters and input as control. We utilized the Genomic Regions Enrichment of Annotations Tool (GREAT 3.0) to analyze the detected peaks, with the parameter “Basal plus extension”, which is proximal: 5 kb upstream, 1 kb downstream, plus Distal: up to 1000 kb. Gene ontology from GREAT output were analyzed by DAVID. KEGG pathways, biological processes, molecular functions, and GAD disease enrichment analysis was carried out using default settings. The HOMER findMotifsGenome.pl script was employed to search for known TRANSFAC motifs and to generate de novo motifs. The intersecBed was used to find overlapped peaks between SMAD3 and TCF21. The filter used to cut off SMAD3 peaks is: fold change> 5 and -logq_value>10. Pooled TCF21 peaks were cut off with three different thresholds, liberal: fold change>5, -logq_value>25; standard: fold change>10, -logq_value>60; stringent: fold change>15, -logq_value>200. The fastq files of SMAD3 ChIPseq in A549 cell lines was extracted from GSM1246721/SRR1014002 by fastq-dump. Similar methods were used in quality control, alignment, peak calling and intersection with SMAD3 peaks in HCASMC. NarrowPeaks is the peaks file call by macs2.1.1 callpeaks module using aligned bam files with default configuration. Aligned reads in Bam file was converted to Bed format by bedtools bamToBed module and then generated .bedGraph file using bedItemOverlapCount. bedGraphToBigWig was used to create the.bigwig file from .bedGraph.
Genome_build: hg19
Supplementary_files_format_and_content: bed and bigwig
|
| |
|
| Submission date |
Jun 04, 2018 |
| Last update date |
Sep 12, 2018 |
| Contact name |
Thomas Quertermous |
| E-mail(s) |
tomq1@stanford.edu
|
| Phone |
650-723-5012
|
| Organization name |
Stanford University
|
| Department |
Medicine Cardiology
|
| Lab |
Quertermous
|
| Street address |
300 Pasteur Drive
|
| City |
Stanford |
| State/province |
CA |
| ZIP/Postal code |
94305 |
| Country |
USA |
| |
|
| Platform ID |
GPL20795 |
| Series (2) |
| GSE115317 |
Coronary artery disease genes SMAD3 and TCF21 promote opposing interactive genetic programs that regulate smooth muscle cell differentiation and disease risk [ChIP-seq] |
| GSE115319 |
Coronary artery disease genes SMAD3 and TCF21 promote opposing interactive genetic programs that regulate smooth muscle cell differentiation and disease risk |
|
| Relations |
| BioSample |
SAMN09348662 |
| SRA |
SRX4161716 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3175516_SMAD3_2.bw |
367.9 Mb |
(ftp)(http) |
BW |
| GSM3175516_SMAD3_2_bam_peaks.narrowPeak.gz |
581.0 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
