|
| Status |
Public on Apr 01, 2018 |
| Title |
ccALD1.rep1 |
| Sample type |
SRA |
| |
|
| Source name |
ccALD1_BMECs
|
| Organism |
Homo sapiens |
| Characteristics |
genotype: cerebral childhood adrenoleukodystrophy (ccALD)
cell type: iPSC-derived Brain microvascular endothelial cells (BMECs)
|
| Treatment protocol |
iPSCs were singularized and seeded on Matrigel in E8 medium with 10 µM Y-27632 (BD Biosciences) (Day -3). On Day 0 differentiation was induced by switching to unconditioned medium (UM): DMEM/F12 (Life Technologies) containing 20% Knockout Serum Replacement (Life Technologies), 1X MEM non-essential amino acids (Life Technologies), 1 mM L-Glutamine (Life Technologies), and 0.1 mM 2-mercaptoethanol (Sigma). Medium was changed every 24 hours. On Day 6, medium was changed from UM to endothelial cell (EC) medium: human endothelial serum free medium (Invitrogen) with 1% platelet-poor plasma derived serum (Biomedical Technologies) supplemented with 20 ng/mL basic fibroblast growth factor (bFGF; R&D Systems) and 10 µM all-trans retinoic acid (RA; Sigma). Medium was not changed for 48 h. On Day 8, cells were detached with Accutase (Invitrogen) and subcultured onto tissue culture polystyrene plates (Corning) coated with a water-based solution of 400 µg/mL collagen IV (Sigma) and 100 µg/mL fibronectin (Sigma). 24 h after subculture, medium was changed to EC medium without bFGF or RA.
|
| Growth protocol |
iPSCs were maintained on Matrigel in E8 medium (Invitrogen).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were detached with trypsin on day 10 of the protocol. Total RNA was extracted using an Rneasy Mini Kit (Qiagen) following the manufacturer's protocol. RNA with a RIN score >8 was used for library generation.
RNA libraries were prepared for sequencing using a TruSeq Stranded mRNA Sample Preparation kit and standard Illumina protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina MiniSeq |
| |
|
| Description |
ccALD1.1
|
| Data processing |
Low quality bases were trimmed using Trimmomatic (enabled with the optional "--qualitycontrol" option and a 3bp sliding-window trimming from the 3' end requiring minimum Q16).
The remaining reads were mapped to hg19 using Tophat2.
The featureCounts program in the R SubRead package was used to generate a transcript abundance file.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimted text file includes read counts
|
| |
|
| Submission date |
Dec 13, 2017 |
| Last update date |
Apr 01, 2018 |
| Contact name |
Jakub Tolar |
| E-mail(s) |
tolar003@umn.edu
|
| Organization name |
University of Minnesota, Twin Cities
|
| Department |
Pediatrics
|
| Street address |
420 Delaware St SE
|
| City |
Minneapolis |
| State/province |
MN |
| ZIP/Postal code |
55455 |
| Country |
USA |
| |
|
| Platform ID |
GPL22790 |
| Series (1) |
| GSE108012 |
RNA-seq of induced brain microvascular endothelial cells from cerebral childhood adrenoleukodystrophy patients and wild-type controls |
|
| Relations |
| BioSample |
SAMN08167384 |
| SRA |
SRX3466675 |
