|
Status |
Public on Feb 14, 2020 |
Title |
HUAEC_ArteryRNAseq_Rep4 |
Sample type |
SRA |
|
|
Source name |
arterial endothelial cells
|
Organism |
Homo sapiens |
Characteristics |
cell type: HUAEC passage: 3 genotype: wildtype chip antibody: n/a
|
Treatment protocol |
HUAEC and HUVEC were used prior to passage 3; for knockdown experiments, HUVEC were transduced with lentiviral vectors expressing a control shRNA or an shRNA targeting NR2F2
|
Growth protocol |
Umbilical cords were collected from UMass Memorial Medical Center and washed with phosphate-buffered saline (PBS) to remove excessive blood. For HUVEC isolation, the vein was cannulated at one end and perfused with 0.1% type 2 collagenase in PBS (Worthington Biochemical), then clamped at both ends, wrapped in aluminum foil and placed in a humidified incubator for 20 minutes at 37°C. Endothelial cells were collected by flushing collagenase solution through the vein with ECCM (EGM Endothelial Cell Growth Medium with SingleQuots Supplement, Lonza). Cells were centrifuged and re-suspended in ECCM, seeded in 0.2% gelatin-coated flasks and allowed to attach overnight at in a humidified incubator (37°C, 5% CO2). Medium was replaced the following day to remove erythrocytes and cells were grown to confluence (2-3 days). The same cords were used for HUAEC isolation by dissecting out arteries, which were washed twice with PBS and perfused with 0.05% Collagenase using a 20-gauge needle (Fisher) attached to a 20 ml syringe. Arteries were clamped on both ends, placed in a beaker containing PBS and incubated for 20 minutes in a 37°C water bath. Detached endothelial cells were collected by flushing the collagenase with EC complete medium followed by centrifugation. For passaging, cells were re-suspended in ECCM and grown for 3-4 days, or until ~80% confluence.
|
Extracted molecule |
polyA RNA |
Extraction protocol |
mRNA was isolated using the NEB PolyA mRNA magnetic bead isolation module RNA-seq libraries were constructed as described in the NEB Poly(A) mRNA magnetic isolation module (#E7490S) and NEB RNA-Seq Library Prep Reagent Set for Illumina (#E6100 L), using mRNA isolated from 5 μg of total RNA with NEB magnetic Oligo d(T)25 beads. In all cases, library quality and concentration were determined using an Agilent bioanalyzer (UMass Medical School Deep Sequencing Core)
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|
|
Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2000 |
|
|
Description |
HUVECvsHUAEC_DEseq2out.csv A26 instrument model unknown by submitter. HiSeq 2000 used as placeholder.
|
Data processing |
basecalls were made with standard Illumina pipeline for indicated instrument for ChIPSeq and Cut&Run, reads were aligned to hg19 using Bowtie2 peak-calling was performed using MACS or MACS2 for RNA-seq, reads were mapped to ENSEMBL annotated transcripts using Tophat2 and differentially expressed genes were identified using EdgeR Genome_build: hg19 Supplementary_files_format_and_content: big wig and bed files
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|
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Submission date |
Mar 15, 2019 |
Last update date |
Feb 14, 2020 |
Contact name |
Nathan D. Lawson |
E-mail(s) |
nathan.lawson@umassmed.edu
|
Organization name |
University of Massachusetts Medical School
|
Department |
Molecular, Cell, and Cancer Biology
|
Street address |
364 Plantation Street
|
City |
Worcester |
State/province |
MA |
ZIP/Postal code |
01605 |
Country |
USA |
|
|
Platform ID |
GPL11154 |
Series (1) |
GSE128382 |
Identification and characterization of artery and vein enhancers in the human genome |
|
Relations |
BioSample |
SAMN11148923 |
SRA |
SRX5527622 |