RNA-seq for Mat2a-KO HCC mice model. Mus musculus

Total RNA was extracted from liver tissues in biological triplicates from 4 weeks Mat2a WT and LKO mice by using illustra RNAspin Mini Kit (GE Healthcare). RNA samples were quantified using Nanodrop and qualified by agarose gel electrophoresis. Illumina kits which include procedures of RNA fragmentation, random hexamer primed first strand cDNA synthesis, dUTP based second strand cDNA synthesis, end-repairing, A-tailing, adaptor ligation and library PCR amplification, were used for RNA-seq library preparation. Finally, the prepared RNA-seq libraries were qualified using Agilent 2000 Bioanalyzer and quantified by qPCR absolute quantification method. The sequencing was performed using Illumina HiSeq 4000. Raw sequencing data generated from Illumina HiSeq 4000 that pass the Illumina chas-tity filter were used for the following analysis. Trimmed reads (trimmed 5,3-adaptor bases) were aligned to reference genome. Base on alignment statistical analysis (map-ping ratio, rRNA/mRNA content, fragment sequence bias), we determine whether the results could be used for subsequent data analysis. If so, the expression profiling, dif-ferentially expressed genes and differentially expressed transcripts were calculated. The novel genes and transcripts were also predicted. Principal Component (PCA), Correlation Analysis, Hierarchical Clustering, Gene Ontology (Go), Pathway Analysis, scatterplots and volcano plots were performed for the differentially expressed genes in R or Python environment for statistical computing and graphics.