Next Generation Sequencing Facilitates Quantitative Analysis of Ctrl-Cas9 and TRIB3-Cas9 cells' Transcriptomes in lymphoma. Next Generation Sequencing Facilitates Quantitative Analysis of Ctrl-Cas9 and TRIB3-Cas9 cells' Transcriptomes in lymphoma

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to analysis the differiational genes and pathways in Ctrl-Cas9 and TRIB3-Cas9 lymphoma cells by using NGS-derived lymphoma transcriptome profiling (RNA-seq). Methods: Ctrl-Cas9 and TRIB3-Cas9 cells' mRNA profiles were generated by deep sequencing, in triplicate, using Illumina GAIIx. The sequence reads that passed quality filters were analyzed at the transcript isoform level with following methods: Alignment by using HISAT2 v2.1, IGV was used to to view the mapping result by the Heatmap, histogram, scatter plot or other stytle, FPKM was then calculated to estimate the expression level of genes in each sample, DEGseq v1.18.0 was used for differential gene expression analysis between two samples with non biological replicates and Function Enrichment Analysis including GO enrichment analysis and KEGG . Conclusions: Our study represents the first detailed analysis of Ctrl-Cas9 and TRIB3-Cas9 cells' transcriptomes, with biologic replicates, generated by RNA-seq technology. The optimized data analysis workflows reported here should provide a framework for comparative investigations of expression profiles. Our results show that NGS offers a comprehensive and more accurate quantitative and qualitative evaluation of mRNA content within a cell or tissue. We conclude that RNA-seq based transcriptome characterization would expedite genetic network analyses and permit the dissection of complex biologic functions. Overall design: Ctrl-Cas9 and TRIB3-Cas9 cells' mRNA profiles were generated by deep sequencing, in triplicate, using Illumina.

研究目的：下一代测序（Next-generation sequencing, NGS）已彻底革新了基于系统视角的细胞通路分析范式。本研究旨在利用NGS衍生的淋巴瘤转录组测序（RNA-seq）技术，分析Ctrl-Cas9与TRIB3-Cas9淋巴瘤细胞中的差异表达基因及调控通路。

研究方法：采用Illumina GAIIx平台对Ctrl-Cas9与TRIB3-Cas9细胞进行三次重复深度测序，获取其mRNA表达谱。对通过质量过滤的测序读段，于转录本异构体水平开展如下分析：使用HISAT2 v2.1进行序列比对；利用IGV以热图、直方图、散点图等形式可视化比对结果；随后计算FPKM以评估各样本中基因的表达水平；采用DEGseq v1.18.0对两个无生物学重复的样本进行差异基因表达分析，并开展功能富集分析，涵盖基因本体（Gene Ontology, GO）富集分析与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）富集分析。

研究结论：本研究首次对采用RNA-seq技术构建、带有生物学重复的Ctrl-Cas9与TRIB3-Cas9细胞转录组进行了详尽分析。本研究报道的优化数据分析流程，可为表达谱的比较研究提供标准化分析框架。研究结果表明，NGS可对细胞或组织内的mRNA含量实现全面且更为精准的定量与定性评估。综上，基于RNA-seq的转录组表征能够加速遗传网络分析，并助力解析复杂的生物学功能。

整体实验设计：采用Illumina平台进行三次重复深度测序，获取Ctrl-Cas9与TRIB3-Cas9细胞的mRNA表达谱。