Computational protocol: The biochemical composition and transcriptome of cotyledons from Brassica napus lines expressing the AtGL3 transcription factor and exhibiting reduced flea beetle feeding

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Protocol publication

[…] Total RNA was extracted from cotyledons of seven-day-old seedlings using an RNAeasy Mini Kit with contaminating gDNA being removed using RNAse-free DNAse™ (Qiagene Inc., ON Canada). RNA samples were quantified and RNA integrity determined using an RNA6000 nano assay in an Agilent 2100 Bioanalyzer™ (Agilent Technologies, Palo Alto, CA USA). RNA library preparation and sequencing were carried out using the Illumina TrueSeq RNA sample preparation platform v.2 with multiplex labeling following the manufacturer’s protocols. Details on cDNA library development, RNA sequencing, and data analysis were identical to those found in Alahakoon et al. []. Cotyledon gene expression changes in the two transgenic hairy leaf lines relative to cv. Westar cotyledon expression were then introduced into MAPMAN for allocation into 36 functional barcode index numbered (BIN) categories []. [...] Cotyledon composition and qRT-PCR data were analyzed with either one-way or two-way ANOVA using a MIXED model in SAS 9.2 [] or a t-test. Assumptions of ANOVA were tested using a Normality test (Shapiro-Wilk), an Equal variance test (Brown Forsythe) and a Levenes test. Means were compared using a Tukey test or pairwise using a Dunn’s method in SAS 9.2, and treatments were declared significant at P ≤ 0.05 and trends declared at P ≤ 0.1. Read counting and statistical analysis of the RNA-seq data were carried out using Cuffdiff in the Cufflinks software package []. […]

Pipeline specifications

Software tools MapMan, Cufflinks
Application RNA-seq analysis