*2.3. Expression Analysis of Putative lncRNAs and Quantitative Reverse Transcription PCR (qRT-PCR) Validation*

RSEM (v1.3.3, Bo Li, Madison, WI, USA) was used to compare the sequenced reads with the transcripts to calculate the gene expression amounts and carry out a differential gene expression analysis. The levels of gene expression were reflected by FPKM values, and a threshold of FPKM > 0.1 was used for the expressed genes. The DESeq package in R software (v3.6.1, R Core Team, Vienna, Austria) was used for pairwise differential expression analysis. The website http://bioinformatics.psb.ugent.be/webtools/Venn/ (accessed on 14 June 2019) was used for Venn diagram drawing [50]. qRT-PCR was performed to validate the expressions of the putative lncRNAs with the same RNA samples as those used for the RNA-seq [51]. The primers are listed in Table S1. GAPDH was chosen as the reference gene. The DDCt method was used to calculate the results [52].
