Computational protocol: Gene Expression Profiling Analysis of Bisphenol A-Induced Perturbation in Biological Processes in ER-Negative HEK293 Cells

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Protocol publication

[…] RNA-seq reads were aligned to the human genome (hg19) using TopHat version 1.3.3 . The reference genome and the corresponding annotation files were downloaded from the UCSC genome browser. Reads alignment was carried out with default parameters except for the following options: “-a 6 -m 2 -i 50–no-novel-juncs”. Only the uniquely mapped reads were used for the subsequent analysis. Reads distribution relative to gene structure was statistically analyzed using Ever-seq (http://code.google.com/p/ever-seq/). BEDTools was used to calculate the coverage of reads along transcripts for each sample.Cuffdiff, a separate program from Cufflinks , was used to calculate gene expression levels and test the statistical significance of expression changes between two samples. Reads Per Kilo base per Million mapped reads (RPKM) was used to evaluate gene expression levels. Pearson correlation coefficient was calculated to measure the overall similarity between transcriptome profiles of the BPA-treated sample and the control sample. Expression changes were measured by log2(fold change) with false discovery rate (FDR)-adjusted p-value as an indicator for statistical significance. Genes with |fold change|> = 1.5 and FDR-adjusted p-value< = 0.05 were defined as the differentially expressed genes as a consequence of BPA exposure.To investigate the functions and involving pathways of genes affected by BPA exposure, we used Ingenuity Pathway Analysis software (IPA, http://www.ingenuity.com) to perform Gene Ontology (GO) and pathway analysis. To assess the biological relationships among these genes, we built molecular interaction networks among them. […]

Pipeline specifications

Software tools TopHat, BEDTools, Cufflinks, IPA
Databases UCSC Genome Browser
Applications RNA-seq analysis, Genome data visualization
Organisms Homo sapiens
Chemicals Cysteine, Estrogens