|
| Status |
Public on Dec 06, 2017 |
| Title |
Diabetic_SAHA_R2 |
| Sample type |
SRA |
| |
|
| Source name |
Diabetic endothelial cells_SAHA
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human aortic endothelial cell
disease state: diabetic
passages: 5-9
|
| Treatment protocol |
Cells were stimulated with DMSO, SAHA (2 μM) or C646 (10 μM) for 12 hours. EP300 knock-down was generated using ON-TARGETplus Human EP300 (2033) siRNA – SMARTpool (L-003486-00-0005, Dharmacon). Cells were cultured in Opti-MEM® (Life Technologies) media with Lipofectamine® RNAiMAX Reagent (13778-075, Life Technologies) with either non-target (ON-TARGETplus Non-targeting Pool, D-001810-10-05, Dharmacon) or 25 nM EP300 siRNA for four hours. Media was replaced with EBM-2 and RNA was harvested after 48 hours.
|
| Growth protocol |
Human aortic endothelial cells (HAEC) from diabetic (CC-2919) and non-diabetic (CC-2535) individuals were maintained in endothelial cell basal growth media-2 (EBM-2, Lonza) with additional EBM-2 growth factors and supplements and with 10% heat-inactivated fetal bovine serum (Gibco). HAECs used in this study were between 5-9 passages.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted in TRIzol (Invitrogen) and purified using the Direct-zolTM RNA MiniPrep kit (cat. no: R2052, Zymo Research) according to the manufacturers instructions. RNA was quantified on the MultiNA bioanalyzer (Shimadzu).
NEBNext® Poly(A) mRNA Magnetic Isolation Module was used to enrich mRNA from 1 μg of total RNA. The NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina® was used to generate barcodes. Libraries were quantified on the MultiNA bioanalyzer (Shimadzu) and pooled to equimolar ratios for sequencing. Cluster generation was performed at a concentration of 12 pM (TruSeq SR Cluster Kit v4-cBot-HS) and the flow cell was run on Illumina HiSeq2500 generating 50 nt reads.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Fastq files were aligned to the hg19 human genome (ensembl GRCh37 release 75) using STAR
The tool featureCounts was used to generate a matrix of read counts per gene
edgeR software was then used to determine differential gene expression
Genome_build: hg19
Supplementary_files_format_and_content: Matrix (text file) of raw reads for each gene (ensembl GRCh37 release 75) for all samples
|
| |
|
| Submission date |
Jan 21, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Assam El-Osta |
| Organization name |
Baker Heart and Diabetes Institute
|
| Lab |
Human Epigenetics
|
| Street address |
75 Commercial Road
|
| City |
Melbourne |
| ZIP/Postal code |
3004 |
| Country |
Australia |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE77108 |
HDAC inhibitor SAHA reverses inflammatory gene expression in diabetic endothelial cells |
|
| Relations |
| BioSample |
SAMN04433006 |
| SRA |
SRX1540358 |
