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Status |
Public on Aug 01, 2022 |
Title |
POR-deficient patient (PORD) iPS-derived BMECs differentuated with 1 uM Retinoic acid [P1_a] |
Sample type |
SRA |
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Source name |
induced pluripoten stem (iPS)-derived brain microvascular endothelial-like cells (iBMECs)
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Organism |
Homo sapiens |
Characteristics |
cell type: induced pluripoten stem (iPS)-derived brain microvascular endothelial-like cells (iBMECs) genotype: POR-deficient patient cell type_source: iPS-derived genotype: POR-deficient (PORD) treatment: 1 uM Retinoic acid
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Treatment protocol |
iPSCs were differentiated into iBMECs as previously described (Lippmann et al., 2014). Briefly, cells were cultured in Nutristem for 3 days after passaging. When cells reached a density of 2x105 cells per well, medium was replaced with 3 ml of unconditioned medium without bFGF (UMF, 1:1 DMEM:F12) supplemented with 20% Knock-out serum-free (KOSR, Gibco), 1% NEAA, 0.5% Glutamax (Gibco), 220 nM β-mercaptoethanol and 1% PSA. On day 6 the medium was replaced with human Endothelial Serum Free medium (ESFM, Gibco) supplemented with 20ng/ml bFGF and 0, 0.25, 1 or 10 µM of RA in DMSO. On day 8, transwells, plastic dishes or coverslips were pre-coated with 100 µg/ml fibronectin (BI) : 400 µg/ml collagen IV (Sigma) and incubated overnight. On the day of seeding the extracellular matrix solution was aspirated and left to dry for 30 mins. iBMECs were harvested in Accutase (Gibco) for 30 mins, then washed with ESFM, counted, and centrifuged. iBMECs were resuspended in ESFM at 1x106 cells/ml and plated on 12 mm with 0.4 µm pore polycarbonate membrane transwell inserts (Corning, cat# 3401); 1 ml of fresh ESFM was supplemented to the bottom chamber and 1 ml of ESFM with cells was applied per transwell. 100 μl were added to 96-wells and 500 μl were added to 24-well plates with coverslips.
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Growth protocol |
The experiments described in this manuscript were approved by the Soroka Medical Center, Beer sheva, under IRB-SCRO protocol 0005-14-SOR. iPSCs were cultured on Matrigel-coated 6-well plates in with daily replacements of Nutristem (Biological Industries, Ltd). Cells were cultured at 37°C and 5% CO2. Cells were passaged weekly using EZ-Lift (Millipore) according to the manufacturer’s instructions.
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Extracted molecule |
total RNA |
Extraction protocol |
Total RNA Purification kit (Norgen Biotek Corp.) NovaSeq6000 SP Reagent kit 100 cycles (Illumina, 20028401)
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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Data processing |
The processed reads were aligned with TopHat , allowing for 5 mismatches to the reference human genome GRCh38, with annotations from Ensembl release 99. Quantification was done with htseq-count. Counts of technical replicates were averaged and merged into a single sample (termed ab) Normalization, quality control plots (PCA, MA-plots) and differential expression analysis were performed using the DESeq2 package Pair-wise comparisons were tested with default parameters using the Wald test, without using the independent filtering algorithm. Significance threshold was taken as p-adj<0.1. In addition, significant genes were further filtered by the log2(fold change) value. This filtering was baseMean-dependent and required a baseMean above 5 and an absolute log2FoldChange higher than 5/sqrt(baseMean) + 0.6. For highly expressed genes this means a requirement for a fold change of at least 1.5, while genes with a very low expression would need a 7-fold change to pass the filtering Assembly: GRCh38, with annotations from Ensembl release 99 Supplementary files format and content: DESeq2 normalized base mean conts per gene per samples after averaging of technical replicates raw counts
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Submission date |
May 08, 2022 |
Last update date |
Aug 01, 2022 |
Contact name |
Inbar Plaschkes |
Organization name |
HUJI
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Department |
The Faculty of Medicine - Ein Kerem
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Street address |
The Hebrew University of Jerusalem - Ein Kerem
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City |
Jerusalem |
ZIP/Postal code |
91120 |
Country |
Israel |
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Platform ID |
GPL24676 |
Series (1) |
GSE202485 |
P450 oxidoreductase (POR) Regulates Barrier Maturation by Mediating Retinoic Acid Metabolism in a Model of the Human BBB |
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Relations |
BioSample |
SAMN28156815 |
SRA |
SRX15206749 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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