Whole Transcriptome Sequencing

Total RNA was extracted, and quantified using Bioanalyzer 2100 (Agilent, CA, United States) with RNA Integrity Number (RIN) > 7.0. Subsequently, a small RNA library and a chain-specific library without ribosomal RNA were established and sequenced. The ribosome-deficient chain-specific library obtain the sequence information of lncRNA but also the sequence information of mRNA and circRNA. The small RNA library provided a miRNA sequence.

Transcripts that overlapped with known mRNAs and those that were shorter than 200 bp were discarded. The coding potential of transcripts was determined using Coding Potential Calculator (CPC) (Kong et al., 2007), Coding-Non-Coding Index (CNCI) (Sun et al., 2013), and Pfam (Punta et al., 2012). Transcripts with CPC scores <−1 and CNCI scores <0 was removed. StringTie and fragments per kilobase of transcript per million fragments (FPKM) (Pertea et al., 2015) were used to determine the levels of mRNAs and lncRNAs, respectively. LncRNAs were analyzed using the Ballgown R package. Those with log₂ fold change (FC) > 1 or log₂ FC <−1 and p value <0.05 were considered differentially expressed (Frazee et al., 2015).