Computational protocol: RNA-Seq Identifies Key Reproductive Gene Expression Alterations in Response to Cadmium Exposure

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Protocol publication

[…] Raw data were mapped to the mouse reference genome (mm10 downloaded from UCSC) using TopHat (version 2.0.3) and RNASEQR (version 1.0.2) software, respectively [, ]. Prebuilt genomic indices were created by bowtie and provided to the alignment software for reads mapping. TopHat removes a few low-quality score reads then aligns the reads that are directly mapped to the reference genome. It then determines the possible location of gaps in the alignment based on splice junctions flanking the aligned reads and uses gapped alignments to align the reads that were not aligned by Bowtie in the first step. Compared with other alignment tools, RNASEQR takes advantages of annotated transcripts and genomic reference sequences to obtain high quality mapping result by the three sequential processing steps. It aligns the RNA-seq sequences to a transcriptomic reference firstly, then detects novel exons and identifies novel junctions using an anchor-and-align strategy finally. The output of Tophat can be the input of Cufflinks, while the output of RNASEQR can be converted as the input for Cufflinks using the script. [...] In order to determine the differentially expressed transcripts within the dataset, we used Cuffdiff, a separate program included in Cufflinks, to calculate expression in two or more samples and test the statistical significance of each observed change in expression between them. Cuffdiff reports numerous output files containing the results of the differential analysis of the samples, including genes and transcripts expression level changes, familiar statistics such as log2-fold change, P values (both raw and corrected for multiple testing), and gene-related attributes such as common name and genome location. The differentially expressed genes were clustered and visualized by TreeView []. Functional annotations of these genes were performed by the DAVID bioinformatics resources [] and WEB-based GEne SeT AnaLysis Toolkit (WebGestalt) []. Downstream enrichment analyses such as TF binding sites in promoter regions and microRNA sites in 3′-UTRs were performed using Expander (version 6.0.5) [], and the miRNA-target gene network was constructed by Cytoscape []. [...] qPCR analyses were performed to validate the results of RNA-seq. The reverse transcription is synthesized using RevertAidTM First Strand cDNA Synthesis Kit from Fermentas according to the manufacturer's instructions. The PCR primers were designed with Primer Premier 5.0 software and β-Actin was used as a reference gene. The primer sequences, melting temperatures, and product sizes are shown in . qPCR was performed on iQ5 Real Time PCR Detection System (Bio-Rad) (Bio-Rad, USA) using SYBR Green Realtime PCR Master Mix (TOYOBO CO., LTD, Japan) as the readout. Three independent biological replicates of the control and cadmium treated groups were included in the analysis and all reactions were carried out in triplicates. Data was analyzed by the 2-ΔΔCT method []. […]

Pipeline specifications

Software tools TopHat, RNASEQR, Bowtie, Cufflinks, TreeViewX, DAVID, WebGestalt, EXPANDER, Primer Premier
Applications Phylogenetics, RNA-seq analysis
Organisms Mus musculus
Diseases Drug-Related Side Effects and Adverse Reactions
Chemicals Cadmium, Testosterone