RNAseq of lung cancer cell A549 cholesterol-3
RNA-seq preparation and RNA-seq data analysis were performed as previously reported (Wang DM,2020). Briefly, total RNA was isolated using the standard RNAiso Plus Protocol and RNA quality was examined. And then RNA samples were purified with magnetic oligo(dT) beads after denaturation and were reverse transcribed into first-strand cDNA. Fragmented DNA samples were end blunted and adenylated at the 3-end. Adaptors were ligated to construct a library. After DNA quantified by Qubit (Invitrogen, NY, USA) and cBot cluster generation, DNA samples were then sequenced by an Illumina HiSeqX Ten SBS instrument (Genergy Bio, Shanghai, China). Raw data were converted into Fastq format. The number of transcripts of each sample was calculated based on the number of fragments per kilobase of transcript per million fragments mapped (FPKM); The FPKM value was calculated by Cuffnorm software for each sample, and the values were log2 transformed. The differential gene transcripts between different samples were calculated using DESeq software. The thresholds for determining differentially expression transcripts are P < 0.05 and absolute fold change  2. Then DETs were chosen for function and signaling pathway enrichment analysis using GO and KEGG database.