|
| Status |
Public on Dec 01, 2023 |
| Title |
RFS2 |
| Sample type |
SRA |
| |
|
| Source name |
Human umbilical cord endothelial cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human endothelial cells
treatment: Static culture
|
| Treatment protocol |
HUVECs were seeded at a density of 500.000 cells per slide in growth medium on gelatin-coated SuperFrost Excell object slides (Thermo Scientific Menzel) and were grown to confluency for 48h. Slides were placed in a custom-designed parallel plate flow chamber within a tissue culture incubator. Wall shear stress was applied (15 dynes/cm2) for 24h using a peristaltic pump (Masterflex LS 7550-30). For static controls, HUVECs were seeded on slides, cultured in the same incubator in the absence of shear stress, and collected at the same time.
|
| Growth protocol |
HUVECs from pooled donors were obtained from PromoCell (C-12208) and cultured in 0,2% gelatin-coated dishes in EGM2 medium supplemented with growth factors (PromoCell, C-22111), 100 units/ml penicillin and 100ug/ml streptomycin (Gibco). HUVECs were used between passages 4 to 6, the medium was refreshed every 48h or 72h, and regularly tested for mycoplasma.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using RNEasy Kits (Qiagen) according to the manufacturer’s instructions. RNA sequencing was performed by the VIB Nucleomics Core (KU Leuven, Belgium). RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyzer 2100 (Agilent).
Per sample, an amount of 250ng of total RNA was used as input. Using the Illumina TruSeq® Stranded mRNA Sample Prep Kit (protocol version: # 1000000040498 v00 October 2017) poly-A containing mRNA molecules were purified from the total RNA input using poly-T oligo-attached magnetic beads. In a reverse transcription reaction using random primers, RNA was converted into first strand cDNA and subsequently converted into double-stranded cDNA in a second strand cDNA synthesis reaction using DNA PolymeraseI and RNAse H. The cDNA fragments were extended with a single 'A' base to the 3' ends of the blunt-ended cDNA fragments after which multiple indexing adapters were ligated introducing different barcodes for each sample. Finally, enrichment PCR was carried out to enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. Sequence libraries of each sample were equimolarly pooled and sequenced on Illumina NovaSeq 6000 (S1 100, 100bp single-end reads (100-8-8-0)) at the VIB Nucleomics Core.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
RFS2
|
| Data processing |
Read mapping: The reads are aligned to the reference genome with STAR 2.5.2b
Quality filtering: With samtools 1.5 we remove reads from the alignment that are non-primary mappings or have a mapping quality ≤ 20
Sorting and indexing: With samtools 1.5 we sort the reads from the alignment according to the chromosomes and index the resulting bam-files.
Assembly: Homo sapiens.Ensembl.GRCh38.88
Supplementary files format and content: Collated count matrix derived from raw data files
|
| |
|
| Submission date |
May 04, 2022 |
| Last update date |
Dec 01, 2023 |
| Contact name |
Sandrine Da Cruz |
| E-mail(s) |
sandrine.dacruz@kuleuven.be
|
| Phone |
+3216320033
|
| Organization name |
VIB-KU Leuven Center for Brain and Disease Research
|
| Department |
KU Leuven Department of Neurosciences
|
| Lab |
Neurophysiology in Neurodegenerative Disorders
|
| Street address |
ON 5 Herestraat 49
|
| City |
Leuven |
| State/province |
Flemish Brabant |
| ZIP/Postal code |
3000 |
| Country |
Belgium |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE202220 |
Shear stress promotes metabolic changes in endothelial cells |
|
| Relations |
| BioSample |
SAMN28091387 |
| SRA |
SRX15153474 |
