Computational protocol: Modeling and rescue of defective blood–brain barrier function of induced brain microvascular endothelial cells from childhood cerebral adrenoleukodystrophy patients

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Protocol publication

[…] Cells were differentiated as described above and detached with trypsin. Total RNA was isolated from WT- and ccALD-iBMECs using an RNeasy Mini Kit (Qiagen) following the manufacturer’s protocol. RNA with a RNA Integrity Number (RIN) score > 8 was used for library generation with the TruSeq Stranded mRNA Sample Preparation kit (Illumina). Paired-end 150 bp length reads were generated using an Illumina MiniSeq. Low quality bases were trimmed using Trimmomatic (enabled with the optional “--qualitycontrol” option and a 3 bp 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 for input into the R package edgeR to identify differentially expressed genes. Ingenuity Pathway Analysis [] was used for network analysis and gene ontology [, ] for pathway analysis. […]

Pipeline specifications

Software tools Trimmomatic, TopHat, Subread, edgeR, IPA
Application RNA-seq analysis
Organisms Homo sapiens
Diseases Adrenoleukodystrophy, Encephalitis, Machado-Joseph Disease
Chemicals Oxygen, Polyethylene Glycols, Fluorescein