Name: Sample 6_p
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Seeds of S. lycopersicum cv. Moneymaker and S. habrochaites LA1777 were germinated in a climate-controlled room at 23°C as described above. Five-day-old germinated seedlings were separated into two groups: half of them were transferred to an identical climate room at 15°C (suboptimal-temperature treatment), whereas the other half remained in the room at 23°C (near-optimal temperature treatment). After 3 days, three replicates of whole roots were collected from both genotypes at the two temperature treatments and immediately frozen in liquid nitrogen. The samples were stored at −80°C until RNA extraction. Seeds of S. lycopersicum cv. Moneymaker and S. habrochaites LA1777 were germinated in a climate-controlled room at 23°C as described above. Five-day-old germinated seedlings were separated into two groups: half of them were transferred to an identical climate room at 15°C (suboptimal-temperature treatment), whereas the other half remained in the room at 23°C (near-optimal temperature treatment). After 3 days, three replicates of whole roots were collected from both genotypes at the two temperature treatments and immediately frozen in liquid nitrogen. The samples were stored at −80°C until RNA extraction. Total RNA from whole roots was extracted by TRIzol reagent according to the manufacturer’s instructions (Sigma-Aldrich). The quantity and quality of total RNA treated with RNase-free DNase I (Thermo Scientific) were determined by an Agilent Technologies 2100 Bioanalyzer and RNA samples with RNA integrity number (RIN) > 8 were used for the RNA sequencing and quantitative RT-PCR analysis. Illumina HiSeq2500 (with v4 kit) and Nextera XT sample preparation kits were used for RNA sequencing and all raw reads were paired-end sequenced. Reads for each of the 12 samples were processed by CLC genomics workbench and were aligned to ITAG-cDNA 2.1 (with S. lycopersicum as reference genome). Read counts of each sample were used to quantify the gene expression. Differential expression analysis was carried out with the negative binomial test of DESeq R package v1.20.0 (Anders and Huber, 2010). The False Discovery Rate (FDR) threshold according the multiple testing correction of Benjamini–Hochberg FDR method (Benjamini and Hochberg, 1995) was applied and genes with an adjusted P-value (Padj ≤ 0.05) and log2-fold change in the ratio between 15 and 23°C of more than 1 and less than -1 defined the significantly differentially expressed genes (DEGs). The Plant MetGenMAP system was used to identify significantly altered pathways and for gene ontology (GO) term enrichment analysis (Joung et al, 2009). Functional annotation was retrieved from Panther (http://pantherdb.org) and SGN tomato (http://solgenomics.net).