Computational protocol: RNA sequencing analysis of gene expression regulated by the transcription factor SlZFP2 during early fruit development

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Protocol publication

[…] Barcoded paired-end sequencing libraries were constructed for the six samples using the TruSeq stranded mRNA kit (RS-122-2101, Illumina) based on the manufactory's instructions. We sequenced the six libraries in one run on an Illumina's Miseq system using the 500-cycles Miseq reagent kit (MS-102-2003). After in-house quality control and removal of the index sequences, the 250-bp paired-end reads were mapped to the tomato reference genome (ITAG2.5) using the Tophat program version 2.0.12 . The mapping was guided by current genome annotation and following parameters were used: –read-mismatches 5 –read-gap-length 3 –read-edit-dist 5 –library-type=fr-firststrand –splice-mismatches 0 and default settings for other parameters. In total, the numbers of read pairs mapped for each replicate were as follows: 1,497,635 (207N, replicate 1; 80.7% mapped), 1,191,334 (207N, replicate 2; 80.6%), 1,279,658 (207N, replicate 3; 83.2%), 573,982 (207, replicate 1; 80.2%), 749,210 (207, replicate 2; 80.2%), and 1,158,542 (207, replicate 3; 80.5%).Uniquely mapped reads were then assembled by Cufflinks program 2.2.1 using following parameters: –GTF-guide –frag-bias-correct –min-frags-per-transfrag 10. Then, differentially expressed genes (adjusted p value of 0.05 or less) were selected by comparison between the RNAi line and its nontransgenic sibling (wild type) using Cuffdiff command in the Cufflinks program with following parameters used: –frag-bias-correct –multi-read-correct –min-alignment-count 10 –FDR 0.05. The analysis identified a total of 2722 differentially expressed genes in the 2 dpa fruits between the SlZFP2 RNAi line 207 and the wild type. After further comparison with the gene list of putative SlZFP2 targets based on bacterial one hybrid screening, 193 genes were identified as direct targets of SlZFP2 in early fruit development. […]

Pipeline specifications

Software tools TopHat, Cufflinks
Application RNA-seq analysis
Organisms Solanum lycopersicum