RNA-sequencing Protocol for Differential Gene Expression

RNA-seq was conducted as previously described (Wang D. et al., 2019 (link)). Total RNA was isolated using TRIzol reagent according to the manufacturer's protocol. Total RNA with RNA integrity number > 7.0 was processed following evaluation using the RNA 6000 Nano LabChip Kit (Agilent, California, USA). Poly (A) RNA was purified from 1 μg of total RNA using Dynabeads Oligo (Salajegheh et al.) 25–61005 (Thermo Fisher, CA, USA) by two rounds of purification and then fragmented under the following conditions: 94°C for 5–7 min. The cleaved RNA fragments were reverse transcribed to create a final cDNA library according to the manufacturer's protocol (mRNA Sequencing Sample Preparation Kit, Illumina). The average insert size for the cDNA library was 300 ± 50 bp. We performed 2 × 150 bp end sequencing (PE150) on an Illumina Novaseq 6000 (LC-Bio Technology Co., Ltd., Hangzhou, China) according to the manufacturer's instructions. Differential expression analysis was performed based on adjusted p-values; volcano plots were prepared to depict fold-change differences in gene expression. Gene Ontology (GO) enrichment and Kyoto Encyclopedia for Genes and Genome (KEGG) analyses of the differentially expressed mRNAs were also conducted. RNA-sequencing data were submitted to Gene Expression Omnibus (GEO) [https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE161737].