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Sample GSM3673426 Query DataSets for GSM3673426
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)
 
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
 
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

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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