Computational protocol: The BEACH Domain Protein SPIRRIG Is Essential for Arabidopsis Salt Stress Tolerance and Functions as a Regulator of Transcript Stabilization and Localization

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Protocol publication

[…] Isolated RNA was checked for RNA integrity (RIN > 7, Agilent 2010 Bioanalyzer [Agilent]) and prepared for Illumina sequencing using the TruSeq RNA sample prep kit versus Illumina. The resulting libraries were sequenced by Illumina HiSeq according to the manufacturers protocol using the Beckmann Coulter service as 12 samples multiplexed on one lane (please find the complete data set under http://www.ncbi.nlm.nih.gov/bioproject/278120).The resulting reads were mapped against the Arabidopsis genome with the TAIR10 gff files for annotation using CLC genomics workbench (Quiagen), and total reads per gene were extracted as the measure for gene expression (). Differential expression was called using the Bioconductor package edgeR []. Read counts were normalized to reads per mapped million, averages were calculated, and log-fold changes were calculated adding one value to avoid division by zero. Gene IDs were functionally annotated using the descriptions from TAIR10 (www.arabidopsis.org) and the MapMan annotation () []. Functional enrichments were calculated based on GO terms using GOrilla [] using the significantly regulated genes as the target group and all genes tested as the background. p-values are calculated using the hypergeometric distribution and corrected with Benjamini Hochberg for multiple hypotheses testing. Additional functional enrichments were calculated for the metabolism-centric MapMan categories using the embedded Wilcoxon sum rank algorithm with Benjamini Hochberg correction []. Using both categorizations for enrichments exploits the strength of the GO annotation (regulation) and the strength of the MapMan categorization (metabolism). […]

Pipeline specifications

Software tools CLC Genomics Workbench, edgeR, MapMan, GOrilla
Databases TAIR
Application RNA-seq analysis
Organisms Arabidopsis thaliana, Homo sapiens, Saccharomyces cerevisiae
Diseases Albinism, Neoplasms