|
| Status |
Public on Jul 07, 2020 |
| Title |
A7_1_GFP+, SAM24327036 Clone A7_2 |
| Sample type |
SRA |
| |
|
| Source name |
sorted hPSC-EC GFP+ (CLDN5+)
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Pluripotent stem-cell derived endothelial cells
sample label: SAM24327036_2
treatment: GFP+ (CLDN5-) sorted
replicate: Clone A7_R1_GFP+
|
| Treatment protocol |
hPSC-ECs were dissociated from plates using Accutase (StemCell Technologies) and filtered before sorting with 30 μm filters (Miltenyi Biotec). Dissociated cells were kept in full media during sorting and sorted in cooled collection tubes with complete EC media supplemented with 20% FBS and 25 mM HEPES. Sorting was performed with BD FACSAria III (BD Biosciences) using 4-way purity precision mode. Reporter cells were sorted by FACS into GFP+ and GFP- populations.
|
| Growth protocol |
Expansion medium consisting of StemPro with 50 ng/mL of VEGFA was used for the first cell division, after which cells were cultured using VascuLife VEGF Endothelial Medium Complete Kit (LifeLine Cell Technology). The final composition of supplements added to the media was as follows: 10% FBS, 4 mM L-glutamine, 0.75 U/mL heparin sulfate, 5 ng/mL FGF-2, 5 ng/mL EGF, 5 ng/mL VEGFA, 15 ng/mL IGF1, 1 µg/mL hydrocortizone hemisuccinate, and 50 µg/mL Ascorbic acid. SB431542 (10 µM) was supplemented to the media.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
After sorting, cells were centrifuged (610 g, 10 min) and lysed in 650 μL RLT lysis buffer (Qiagen) + β-mercaptoethanol (Sigma-Aldrich, 1%) and subsequently vortexed for 1 min at room temperature before being snap frozen. RNA isolation was performed using a RNAeasy micro kit including DNaseI digestion (Qiagen).
Total RNA was subjected to oligo (dT) capture and enrichment, and the resulting mRNA fraction was used to construct cDNA libraries. Sequencing was performed on an Illumina HiSeq 4000 platform using the standard protocol (TruSeq Stranded Total RNA Library, Illumina) that generates approximately 30 million reads of 50 base pairs per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
GFP+ cells represent CLDN5+
|
| Data processing |
Basecalling and sample de-multiplexing was performed with CASAVA-1.8.4 and default parameters
Reads were quality check with fastqc v0.11.5 default parameters
Reads for each sample/replicate were mapped against the human genome assembly using STAR read aligner v2.5.2a
RefSeq annotated genes were quantified with samtools v1.5 and in-house scripts
Genome_build: hg38
Supplementary_files_format_and_content: Read counts per gene
|
| |
|
| Submission date |
Dec 19, 2019 |
| Last update date |
Jul 07, 2020 |
| Contact name |
Jitao David Zhang |
| E-mail(s) |
jitao_david.zhang@roche.com
|
| Phone |
+41616886251
|
| Organization name |
F. Hoffmann-La Roche
|
| Department |
Roche Pharmaceutical Research and Early Development, Roche Innovation Center Basel
|
| Lab |
Pharmaceutical Sciences
|
| Street address |
Grenzacherstrasse 124
|
| City |
Basel |
| ZIP/Postal code |
4070 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE142321 |
Genome wide RNA-sequencing of sorted human pluripotent stem-cell derived endothelial cells with CLDN5-GFP reporter (GFP+ and GFP-) |
|
| Relations |
| BioSample |
SAMN13632933 |
| SRA |
SRX7415560 |
