Transcriptome changes of tomato internode elongation induced by far-red light 3 91 Table 3.1. Primer sequences adapted in library preparation. Primer name Sequence PE1-lig 5’-CACTCTTTCCCTACACGACGCTCTTCCGATCT-3’ ILL-lig 5’-P-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC-3’ EnrichS1 5’-AATGATACGGCGACCACCGA-3’ EnrichS2 5’-CAAGCAGAAGACGGCATACGA-3’ Biotin 5’-ACAGGACATTCGTCGCTTCCTTTTTTTTTTTTTTTTTTTT-3’ 3.5.5 Sequencing and alignment Barcoded libraries were sequenced on the High Output : 1 x 75 bp Illumina NextSeq500 at USEQ (Utrecht Sequencing Facility). The initial read quality assessment with FastQC, followed by sequence trimming using TrimGalore (https://github.com/FelixKrueger/ TrimGalore/blob/master/Docs/Trim_Galore_User_Guide.md), rRNA removal via SortMeRNA, read mapping and read-group annotation utilizing STAR (Dobin et al., 2013), alignment quality control with RSeQC (Wang et al., 2012) and Preseq ( https:// smithlabresearch.org/software/preseq/), PCR duplicate detection employing Sambamba MarkDup (Tarasov et al., 2015) , and gene expression and biotype quantification through featureCounts (Liao et al., 2014). The sequences were mapped against ITAG4.1 annotation of tomato reference genome SL4.1 https://solgenomics.net/organism/ Solanum_lycopersicum/genome. 3.5.6 Differential expression analysis We adapted the DE analysis method from literature (Kajala et al., 2021). Libraries with at least 500,000 mapped raw exon counts were selected for further analysis. First, we wanted to identify the differentially expressed genes (DEGs) for various comparisons. Raw counts were firstly converted to count per million (CPM) using with edgeR package (Robinson et al., 2009). Genes with CPM > 0.5 in at least one sample for all 4 biological replicates were kept for the analysis. CPM values were quantile normalized with the voom function (Law et al., 2014). Principal component analysis (PCA) was conducted to distinguish the separation of samples by timepoint, treatment and cultivar with the ggplot2 package (Wickham, 2009) in R.4.1.3 (R Development Core Team, 2010). DEGs were detected with the limma R package (Ritchie et al., 2015). The data underwent a Log2 transformation, and a significance threshold of FDR-adjusted p-value (adj.P.Val) ≤ 0.15 was applied to identify differentially expressed genes. The adjusted P value list of DEGs were obtained for further analysis. Venn plots were generated by DEGs using imageGP website (Chen et al., 2022b).
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