2.7 Illumina RNA sequencing and data analysis
Young leaves and shoot tips from four-week-old WT and CRIR1 OE lines grown under normal and cold-treated (4°C, 24h) conditions were harvested. Two biological replicates consisting of six independent plants for each sample were conducted. The total RNA isolation, whole transcriptome libraries preparation, and deep sequencing were performed by Gene Denovo Biotechnology Co., Ltd. (Guangzhou, China). Total RNA from each sample was extracted using Trizol reagent (Life Technologies). According to the manufacturer’s instructions, whole transcriptome libraries were constructed using Next® Ultra™ RNA Library Prep Kit (New England Biolabs). The libraries were sequenced initially on an Illumina NovaSeqTM 6000 instrument that generated paired-end reads of 150 nucleotides. After removing the adapters and low-quality bases, clean reads were aligned to the cassava genome assembly with HISAT (Kim et al., 2015), then transcript construction using Stringtie (Pertea, Pertea, Antonescu, Chang, Mendell & Salzberg, 2015). The FPKM value was calculated for each unigene using RSEM (Li & Dewey, 2011). The differentially expressed genes (DEGs) were identified by using DESeq2 (Love et al., 2014), with FDR < 0.05. Gene Ontology (GO) enrichment analyses were conducted using AgriGO (Tian, Liu, Yan, You, Yi, Du, Xu & Su, 2017). The RNA-seq data have been deposited to the National Center for Biotechnology Information (NCBI) under the accession number PRJNA522309.