Transcriptome RNA-seq process

Total RNA was extracted via a TRK1001 Total RNA Purification Kit (LC Science, Houston, TX) according to the manufacturer’s protocol [93]. The total RNA amount and purity were determined using a Bioanalyzer 2100 and RNA 6000 Nano LabChip Kit (Agilent, CA, USA), with RNA values > 7.0. After the total RNA was quantified, the eukaryotic mRNA was enriched by attachment to Oligo (dT) magnetic beads. The extracted mRNA was randomly broken into short fragments by fragmentation buffer, and the fragmented mRNA was used as a template to synthesize a strand of cDNA with six-base random primers (random hexamers), double-stranded cDNA synthesis in buffer, dNTPs, RNaseH and DNA Polymerase I [94]. AMPure XP beads were used to purify the double-stranded product, both T4 DNA polymerase and Klenow DNA polymerase were used to repair attachment of the sticky end of the DNA to the blunt end, A bases and linkers were added to the 3′ end, AMPureXP beads were used for fragment selection, and PCR amplification was ultimately performed to increase the final sequencing library. After the library was quantified, it was generated by an Illumina HiSeq 4000 instrument, and the sequencing read length was double-ended 2*150 bp (PE150) [93]; low-quality reads were removed. The raw sequence data are available under GEO Series accession number {"type":"entrez-geo","attrs":{"text":"GSE114968","term_id":"114968"}}GSE114968. Differential expression and functional analysis of the genes were performed. The determination of the different gene expression levels was based on the above data analysis program, with HISAT software used to compare the sequencing data to the NCBI (https://www.ncbi.nlm.nih.gov/genome/?term=Beta+vulgaris) sugar beet reference genome. The transcripts were assembled using multiple alignments. Finally, R was used to graphically display the data results generated by Ballgown. Functional analysis of the DEGs included the use of GOseq for GO enrichment analysis and KOBAS for KEGG signaling pathway enrichment analysis. The expression levels of all transcripts were calculated by StringTie and Ballgown. StringTie was also used to determine mRNA expression levels by calculating the fragments per kilobase of transcript per million mapped reads (FPKM) [94]. Differentially expressed mRNAs and genes were selected by the R package Ballgown according to the criteria of log 2 (fold change) > 1 or log 2 (fold change) < − 1 and were statistically significant when P < 0.05 [95].