|
| Status |
Public on Sep 11, 2018 |
| Title |
HCASMC scrambled siRNA_S18 |
| Sample type |
SRA |
| |
|
| Source name |
Cultured coronary artery smooth muscle cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: coronary artery smooth muscle cells
culture method: Grown in serum
knockdown: scrambled siRNA
|
| Treatment protocol |
SMAD3 (s8401 and s8402) silencer select siRNAs were purchased from Life Technologies. siRNA transfection was performed using Lipofectamine RNAiMAX (Life Technologies). For each well treated with the SMAD3 siRNA or scrambled control (Life technologies, #4390843), the final concentration was 20 nM. HCASMCs were seeded in 6 well plates and grown to 75% confluence before siRNA transfection. HCASMCs were transfected with the SMAD3 siRNA or scrambled control for 12 hours and subsequently collected and processed for RNA isolation after 48 hrs of transfection using the RNeasy kit (Qiagen).
|
| Growth protocol |
Primary human coronary artery smooth muscle cells (HCASMCs) were purchased from three different manufacturers, PromoCell, Lonza and Cell Applications at passage 2 and were cultured in smooth muscle cell basal media along with hEGF, insulin, hFGF-B and fetal bovine serum (FBS) (Lonza # CC-3182) according to the manufacturer’s instructions. HCASMCs between passages 5-8 were used for all the experiments.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA for all samples was extracted using the RNAeasy mini kit (Qiagen). HCASMC RNA (500 ng) were reverse transcribed using the High capacity RNA-to-cDNA Synthesis kit (Applied Biosystems).
Three experimental and three control samples were generated, RNA was converted to cDNA library as per workflow at Novogene, and sequenced on a HiSeq 4000 machine, 125 bp paired end reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
SCR-2_S18
|
| Data processing |
Reads were processed using rnaSeqFPro, a workflow for full processing of RNASeq data starting from fastq files. In brief, the quality control was performed using FastQC, mapping to the human genome hg19 was performed using STAR second pass mapping to increase the percentage of mapped reads, and counting was done with featureCounts using GENCODE gtf annotation. Next, rnaSeqFPro performed differential analysis using DESeq2, conducted principal component analysis and hierarchical clustering using standard R functions, plotPCA and heatmap.2 and generated graphs using gglot2. DESeq2 gave 493 differentially expressed (DE) genes (FDR £ 0.05).
Genome_build: hg19
Supplementary_files_format_and_content: text file with raw counts.
|
| |
|
| Submission date |
Jun 04, 2018 |
| Last update date |
Nov 24, 2019 |
| 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 |
GPL20301 |
| Series (2) |
| GSE115318 |
Coronary artery disease genes SMAD3 and TCF21 promote opposing interactive genetic programs that regulate smooth muscle cell differentiation and disease risk [RNA-seq] |
| GSE115319 |
Coronary artery disease genes SMAD3 and TCF21 promote opposing interactive genetic programs that regulate smooth muscle cell differentiation and disease risk |
|
| Relations |
| Reanalyzed by |
GSE140898 |
| BioSample |
SAMN09348204 |
| SRA |
SRX4161711 |
