Computational protocol: Ligand-mediated cytoplasmic retention of the Ah receptor inhibits macrophage-mediated acute inflammatory responses

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Protocol publication

[…] Total RNA was isolated from primary peritoneal macrophages as previously described using TRI reagent (Sigma, St. Louis, MO) in combination with the RNeasy purification kit (Qiagen, Hilden, Germany). Briefly, 70% ethanol was added to the aqueous phase after phase separation and transferred to RNeasy purification columns. RNA was extracted according to manufacturer’s instructions. RNA quality was assessed on the Agilent Bioanalyzer using the RNA Nano assay. The RNA samples were then Poly-A selected and barcoded libraries were made from each sample using the Illumina TruSeq Stranded mRNA Library Prep Kit following the manufacturer's protocol. We then performed qPCR (Kapa kit) on all libraries to determine concentration. We made an equimolar pool of all barcoded libraries and sequenced the pool on the Illumina HiSeq 2500 in Rapid Run mode using 150 nt single read sequencing at the Genomics Core Facility (The Pennsylvania State University). RNA-seq reads were aligned to the Mus musculus genome (mm10, RefSeq genes) using TopHat version 2.0.13 with default parameters. Alignment results are given in . Reads mapping to genes were counted using HTseq-count version 0.5.4p3 with parameters ‘-s no -a 10’ . […]

Pipeline specifications

Software tools TopHat, HTSeq
Application RNA-seq analysis
Organisms Mus musculus
Chemicals Uric Acid