Computational protocol: Transcriptomic analysis of salt stress responsive genes in Rhazya stricta

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Protocol publication

[…] Filtered reads were aligned with up to two mismatches to the R. stricta reference nuclear genome [] available at NCBI (accession no. MEJB00000000.1). The remaining unmapped sequences were re-aligned against the contigs and collectively de novo assembled using the Trinity RNA-Seq Assembly package (r2013-02-25) following the methods described in Zhang et al. []. RSEM v1.1.6, an RNA-Seq quantification tool, was used to estimate the relative abundances and expected read counts for the transcripts. RSEM uses the Bowtie aligner (Bowtie v0.12.1) to map the reads against the transcripts. Transcript quantification of the reference-aligned as well as the de novo assembled reads was performed with RSEM, which allowed for the assessment of transcript abundances based on the mapping of RNA-Seq reads to the assembled transcriptome. Expected read counts were input into differential expression (DE) analysis using EdgeR (version 3.0.0, R version 2.1.5). The median value from biological replicates was used as the common dispersion factor for DE analyses. DE transcripts were annotated and KEGG pathway analyses were performed using blast-2-GO software (version 2.3.5, http://www.blast2go.com/) and GO terms were obtained with the default parameters. Blastx was performed against the NCBI non-redundant protein database with an E-value cut off of 1e-5. To identify clusters with functional enrichment, we determined a significant Pearson correlation through permutation analysis []. The resulting clusters were refined by visual inspection and analyzed for GO term enrichment using Blast2GO (http://www.blast2go.com/). We also clustered the RPKM data to provide a representation of absolute abundance of the transcripts. [...] Validation of the RNA-Seq data was performed via semi-quantitative RT-PCR for randomly selected transcripts of genes encoding PPR proteins that were upregulated at the 12 h time point. The actin gene of R. stricta was used as the unregulated housekeeping gene. Primers were designed with Netprimer software (http://www.premierbiosoft.com/netprimer/index.html) using five criteria: length ~26 basepairs, GC content ~50%, minimal secondary structure, comparable annealing temperatures (55–56°C) of the primer pairs and PCR products of ~300–400 bp. […]

Pipeline specifications

Software tools Trinity, RSEM, Bowtie, edgeR, Blast2GO, BLASTX, NetPrimer
Databases KEGG PATHWAY
Applications RNA-seq analysis, qPCR
Organisms Rhazya stricta
Diseases Neoplasms
Chemicals Chlorophyll, Flavonoids, Oxygen, Sodium Chloride, Uridine Diphosphate