RNA sequencing and data analysis

HepG2 cells were transfected with si-NEAT1 or si-NC in duplicate. Forty-eight hours after transfection, total RNA was isolated from the cells using TRIzol (Thermo Fisher Scientific) according to the manufacturer’s protocol. RNA purity was assessed using an ND-1000 Nanodrop instrument. The A260:A280 and A260:A230 ratios of each RNA sample exceeded 1.8 and 2.0, respectively. RNA integrity was evaluated using an Agilent 2200 TapeStation (Agilent Technologies, Santa Clara, CA, USA), and the RNA integrity number evaluation of each sample exceeded 7.0. Briefly, mRNAs were isolated from the total RNA and fragmented to a size of approximately 200 bp. Subsequently, second strand of cDNA was synthesized, followed by adaptor ligation and enrichment with a low cycle according to the instructions of the TruSeq^® RNA LT/HT Sample Prep Kit (Illumina, San Diego, CA, USA). The purified library products were evaluated using the Agilent 2200 TapeStation and Qubit^® 2.0 (Thermo Fisher Scientific). The samples were then diluted to 10 pM for cluster generation in situ on the HiSeq 2500 pair-end flow cell, followed by sequencing (2×100 bp) on HiSeq 2500. The transformed data of the two groups were compared using Welch’s t-test. The threshold for up- and downregulated genes was defined as a fold change >2.0 and a P-value <0.05. Geneontology and Kyoto Encyclopedia of Genes and Genomes analyses were employed to determine the roles of these differentially expressed mRNAs.