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.