BW_48h_1.R1
Liver samples were subjected to total RNA extraction utilizing TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA quality was determined by a 5300 Bioanalyser (Agilent) and quantified using an ND-2000 (NanoDrop Technologies). Only high-quality RNA samples (OD260/280=1.8~2.2, OD260/2302.0, RIN6.5, 28S:18S1.0, >1 g) were used to construct the sequencing library. RNA purification, reverse transcription, library construction, and sequencing were performed at Shanghai Majorbio Biopharm Biotechnology Co., Ltd. (Shanghai, China) according to the manufacturers instructions (Illumina, San Diego, CA). The RNA-seq transcriptome library was prepared following Illumina Stranded mRNA Prep, Ligation from Illumina (San Diego, CA) using 1 g of total RNA. Briefly, messenger RNA was isolated according to the polyA selection method by oligo(dT) beads and then fragmented by fragmentation buffer. Second, double-stranded cDNA was synthesized using a SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA) with random hexamer primers (Illumina). Then, the synthesized cDNA was subjected to end repair, phosphorylation, and A base addition according to Illuminas library construction protocol. Libraries were size selected for cDNA target fragments of 300 bp on 2% Low Range Ultra Agarose followed by PCR amplification using Phusion DNA polymerase (NEB) for 15 PCR cycles. After quantification by Qubit 4.0, the paired-end RNA-seq sequencing library was sequenced with a NovaSeq 6000 sequencer (2  150 bp read length).