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Sample GSM3318653

Query DataSets for GSM3318653

Status

Public on Mar 23, 2020

Title

EC_2

Sample type

SRA

Source name

Endothelial cell (EC) differentiation from human embryonic stem cells (hESC)

Organism

Homo sapiens

Characteristics

cell type: day 7 selected endothelial cells (CD144+CD31+)
replicate: replicate 2

Treatment protocol

Cell populations were dissociated by enzymatic digestion. Harvested cells were washed in PBS and incubated with specific antibodies (BD Bioscience). FACS sorting was performed using anti-human CD326, anti human-CD56, anti human-CD31, and anti human-CD144 by FACSAria I or Aria III cell sorter (BD Biosciences). Sorted cell populations were taken forward for RNA extraction.

Growth protocol

A previously published protocol method was employed to generate endothelial cells from H9 human embryonic stem cells (Boulberdaa et al, 2016 Mol Ther). Briefly, hESCs, were dissociated into single cells using Tryple Select (Life Technologies). 1 × 10^4 cells were seeded into a 5% Pluronic F-127 (Sigma-Aldrich) coated 96- well round bottom microplates to generate embryoid bodies (EBs). The plates were centrifuged at 300 g for 3 minutes. Cells were cultured in Stemline II Hematopoietic expansion medium with the addition of 5 ng/mL Activin A (Peprotech), 10 µmol/L Y-27632 (Millipore), 10 ng/mL vascular endothelial growth factor (VEGF) (Peprotech), and 10 ng/mL bone morphogenetic protein 4 (BMP4) R and D Systems, Minneapolis. After 2 days, the EBs were supplemented Stemline II Hematopoietic expansion medium containing 10 ng/mL VEGF, 70 ng/mL Wnt3A, 140 ng/mL BMP4 and 35 ng/mL Activin A. On day 3, EBs were transferred to 0.1% gelatine-coated 6 well culture dishes (Sigma-Aldrich) and resuspended in endothelial growth medium-2 (EGM-2) (Lonza). The media was supplemented with 50 ng/ml of VEGF and the accompanying bullet kit, minus the VEGF and FBS supplement. Cells were maintained until day 7, when they were harvested.

Extracted molecule

total RNA

Extraction protocol

Total RNA was isolated using the miRNeasy kit (Qiagen)
Ribosomal-depleted stranded libraries were prepared by Beckman Coulter Genomics and sequenced with an Illumina HiSeq (paired end 2 x 100bp).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

Fastq files obtained from Beckman Coulter Genomics
Mapping was performed by Beckman Coulter Genomics on the human genome reference sequence GRCh38, using Tophat version 2.0.10 in conjunction with Bowtie version 1.0.0. Tophat was provided with a transcriptome reference generated from the Ensembl annotation of GRCh38. HTSeq was used to obtain a read count for all genes of the GRCh38 ENSEMBL annotation.
The average gene expression value in each population, given as Fragments Per Kilobase of transcript per Million map read (FPKM), was obtained using Cufflinks.
Genome_build: GRCh38
Supplementary_files_format_and_content: txt file with FPKM

Submission date

Aug 03, 2018

Last update date

Mar 23, 2020

Contact name

Julie Rodor

E-mail(s)

Julie.Rodor@ed.ac.uk

Phone

+44 (0)7580200884

Organization name

UNIVERSITY OF EDINBURGH