Next Generation Sequencing Facilitates Quantitative Analysis of TICs with or without H19 knockdown.

The goals of this study are to compare mRNA and miRNA profiling between Tumor Initiating hepatoCytes (TICs) with or without H19 knockdown by shRNA. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 16,014 transcripts in the retinas of WT and Nrl-/- mice with BWA workflow and 34,115 transcripts with TopHat workflow. RNA-seq data confirmed stable expression of 25 known housekeeping genes, and 12 of these were validated with qRT–PCR. RNA-seq data had a linear relationship with qRT–PCR for more than four orders of magnitude and a goodness of fit (R2) of 0.8798. Approximately 10% of the transcripts showed differential expression between the WT and Nrl-/- retina, with a fold change =1.5 and p value <0.05. Altered expression of 25 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Hierarchical clustering of differentially expressed genes uncovered several as yet uncharacterized genes that may contribute to retinal function. Data analysis with BWA and TopHat workflows revealed a significant overlap yet provided complementary insights in transcriptome profiling. Conclusions: Our study showed that knockdown of H19 in TICs led to increased expression of TP53 and CDKN1A/p21 target genes, increased expression of genes which are normally suppressed by Akt, and decreased expression of Src target genes Overall design: Methods: mRNA and miRNA profiles of TICs with or without H19 knockdown were generated by deep sequencing using Illumina HiSeq. For mRNA, reads were trimmed and mapped to the mouse GRCm38 reference genome using the STAR aligner v.2.5.2b. Totally, 40M unique mapped reads for each samples were used for differential gene expression analysis using DESeq2 package. For miRNA, 6-8 M reads (with length of 15 to 31 nucleotides) per sample were retained for subsequent analysis. To annotate the small RNAs, trimmed reads were searched against miRBase 21.