Computational protocol: Rifampin modulation of xeno‐ and endobiotic conjugating enzyme mRNA expression and associated microRNAs in human hepatocytes

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Protocol publication

[…] RNA‐seq library construction, EZBead preparation, and NextGen sequencing were performed using standard methods as described previously and used to measure UGT, SULT, NAT, TPMT, and GST mRNAs and compared to vehicle control (0.1% methanol). UGT1A genes were identified and quantified by unique exons 1 as exons 2‐5 are shared across this gene subfamily. The RNA‐Seq data analysis included quality assessment and sequence alignment prior to differential gene expression analysis as described previously. In brief, SOLiD Instrument Control Software and Experiment Tracking Software were used for read quality recalibration. Each sequence was scanned for low‐quality reads and any read length of less than 35 bases was discarded to effectively eliminate low‐quality reads while retaining high‐quality regions. BFAST was used as the primary sequence alignment algorithm employing a TopHat‐like strategy to align sequencing reads that crossed splicing junctions. Sequence reads were aligned to a filtering index to exclude sequences that were not of interest (eg, repeats and ribosomal RNA). Analyses were restricted to uniquely aligned sequences with 2 or less mismatches. Differentially expressed genes were identified using edgeR following exclusion of genes with less than 1 read per million mappable reads in more than half of samples. A generalized linear model considering the effects of individual donors as a random effect was used to identify gene expression levels directly affected by rifampin treatment. The P‐values were calculated for each gene and Benjaminin and Hochberg's algorithm was used to control the false discovery rate. Data reported in the primary tables and figures only for genes up‐ or downregulated >1.25‐fold by rifampin and P < .05. Clustering of mRNA expression changes and hepatocyte donors depicted in dendrograms were determined, using Euclidian distances and the complete linkage clustering method. Data visualization and hierarchical cluster analysis were performed with R software (build 3.2.3) and R Studio (v. 0.99.491), using the gplots and ggplots2 packages. […]

Pipeline specifications

Software tools BFAST, TopHat, edgeR, gplots
Organisms Homo sapiens