|
| Status |
Public on Mar 05, 2020 |
| Title |
Glucose GEnC derived EVs1 [RNA-seq] |
| Sample type |
SRA |
| |
|
| Source name |
Podocytes
|
| Organism |
Homo sapiens |
| Characteristics |
podocyte treatment: GEnC derived EVs
genc treatment: Glucose
|
| Treatment protocol |
GENCs were incubated in microvascular cell growth kit media supplemented with 5% exosome depleted FBS. GEnCs were either unstimulated (steady state, SS), or stimulated with glucose (Glu, 30mmol), or puromycin aminonucleoside (PAN, 100ug/mL) for 24hr. Cell supernatants were spun at 300g for 10 min, then 2,000g for 15min, and then ultracentrifuged for 10,000g for 30min to remove cell debris and large EVs. Equal supernatant volumes were then ultracentrifuged at 100,000g for 70min at 4℃. EV pellets were resuspended in 300uL of podocyte media plus 5% exosome depleted FBS. 2x10^5 podocytes were untreated (UT) or treated with EVs from GEnCs from SS, GLu or PAN conditions for 24hr.
|
| Growth protocol |
GEnCs were incubated at 33℃ in 5% CO2 to proliferate in microvascular cell growth kit, then thermoswitched at 37℃, 5% CO2 in attachment factor coated plates. Podocytes were incubated at 33℃ in 5% CO2 in RPMI supplemented with 10% PS, 25% FCS, 5% L-glutamine, and 5% ITS. Cells were induced to differentiate at 37℃ in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA from 2x10^5 podocytes incubated for 24h with EVs derived from GEnCs was isolated using the Total RNA Purification Kit (Norgen Biotec) according to the manufacturer's instructions.
Libraries were prepared using the Nugen Ovation Human FFPE RNA-Seq Library Systems kit
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Reads were trimmed to remove adapter sequences using Cutadapt v1.16
Reads were aligned to the human b37 genome using STAR v2.5.3a. Ensembl v75 gene annotations were provided to STAR to improve the accuracy of mapping.
Quality control on both raw reads and adaptor-trimmed reads was performed using FastQC
featureCounts v1.15.29 was used to count the number of mapped reads to each gene.
Significantly differential expressed genes with p-value < 0.05 and absolute fold change > 1.5 were detected by DESeq2 (v1.18.1)
Genome_build: human b37 genome
|
| |
|
| Submission date |
Apr 16, 2019 |
| Last update date |
Mar 05, 2020 |
| Contact name |
Danielle L Michell |
| E-mail(s) |
danielle.michell@vumc.org
|
| Organization name |
Vanderbilt Univ. Medical Center
|
| Department |
Department of Medicine
|
| Lab |
312 Preston Research Bldg
|
| Street address |
2220 Pierece Ave
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37232 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE129883 |
RNA-seq of podocytes treated with glomerular endothelial cell vesicles |
| GSE129892 |
Glomerular endothelial derived vesicles mediate podocyte dysfunction via extracellular transfer of miRNA-200c-3p |
|
| Relations |
| BioSample |
SAMN11435814 |
| SRA |
SRX5694005 |
