design: Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library ordTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification.The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries will be pooled and sequenced on Illumina platforms, according to effective library concentration and data amount.The clustering of the index-coded samples was performed according to the manufacturers instructions. After cluster generation, the library preparations were sequenced on an Illumina platform and paired-end reads were generated.Raw data (raw reads) of fastq format were firstly processed through in-house perl scripts. In this step, clean data (clean reads) were obtained by removing reads containing adapter, reads containing ploy-N and low quality reads from raw data. At the same time, Q20, Q30 and GC content the clean data were calculated. All the downstream analyses were based on the clean data with high quality.Reference genome and gene model annotation files were downloaded from genome website directly. Index of the reference genome was built using Hisat2 v2.0.5 and paired-end cleanReads were aligned to the reference genome using Hisat2 v2.0.5. We selected Hisat2as the mapping tool for that Hisat2 can generate a database of splice junctions based on the gene model annotation file and thus a better mapping result than other non-splice mapping tools.Feature Counts v1.5.0-p3 was used to count the reads numbers mapped to each gene. And then FPKM of each gene was calculated based on the length of the gene and reads count mapped to this gene. FPKM, expected number of Fragments Per Kilobase of transcript, sequence per Millions base pairs sequenced, considers the effect of sequencing depth and gene length for the reads count at the same time, and is currently the most commonly used method for estimating gene expression levels.(For DESeq2 with biological replicates) Differential expression analysis of two conditions/groups (two biological replicates per condition) was performed using the DESeq2R package (1.20.0). DESeq2 provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochbergs approach for controlling the false discovery rate . Genes with an adjusted P-value <=0.05found by DESeq2 were assigned as differentially expressed. (For edgeR without biological replicates) Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package throughone scaling normalized factor. Differential expression analysis of two conditions was performed using the edgeR R package (3.22.5). The P values were adjusted using the Benjamini & Hochberg method. Corrected P-value of 0.05 and absolute fold change of 2 were set as the threshold for significantly differential expression. Gene Ontology (GO) enrichment analysis of differentially expressed genes was implemented by the cluster Profiler R package, in which gene length bias was corrected. GOterms with corrected P values less than 0.05 were considered significantly enriched by differential expressed genes. KEGG is a database resource for understanding high-level functions and 3utilities of the biological system, such as the cell, the organism and the ecosystem, from molecular-level information, especially large-scale molecular datasets generated by genome sequencing and other high-through put experimental technologies(http://www.genome.jp/kegg/). We used cluster Profiler R package to test the statistical enrichment of differential expression genes in KEGG pathways. The Reactome database brings together the various reactions and biological pathways of human model species. Reactome pathways with corrected Pvalue less than 0.05 were considered significantly enriched by differential expressed genes. The DO (Disease Ontology) database describes the function of human genes and diseases. DO pathways with corrected P value less than 0.05 were considered significantly enriched by differential expressed genes. The DisGeNET database integrates human disease-related genes. DisGeNET pathways with corrected P values less than 0.05 were considered significantly enriched by differential expressed genes. We used cluster Profiler software to test the statistical enrichment of differentially expressed genes in the Reactome pathway, the DO pathway, and the DisGeNET pathway.Gene Set Enrichment Analysis (GSEA) is a computational approach to determine if apre-defined Gene Set can show a significant consistent difference between two biological states. The genes were ranked according to the degree of differential expression in the two samples, and then the predefined Gene Set were tested to see if they were enriched atthe top or bottom of the list. Gene set enrichment analysis can include subtle expression changes. We use the local version of the GSEA analysis tool http://www.broadinstitute.org/gsea/index.jsp, GOKEGGReactomeDOand DisGeNETdata sets were used for GSEA independently. GATK (v4.1.1.0) software was used to perform SNP calling. Raw vcf files were filtered with GATK standard filter method and other parameters (cluster:3; WindowSize:35; QD<2.0;FS > 30.0; DP < 10. AS analysisAlternative Splicing is an important mechanism for regulate the expression of genes and the variable of protein. rMATS(4.1.0) software was used to analysis the AS event. PPI analysis of differentially expressed genesPPI analysis of differentially expressed genes was based on the STRING database, which known and predicted Protein-Protein Interactions.Fusion gene refers to the chimeric gene formed by the fusion of all or part of the sequences of two genes, which is generally caused by chromosome translocation, deletion and other reasons. We used Starfusion software (1.9.0) to detect genes that are fused. Star-fusion is a software package uses fusion output results of STAR alignment to detect fusion transcripts, including SATR alignment and SATRfusion. SATR-fusion filter was used to correct the predicted results of Star-fusion to ensure the accuracy of the results.