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摘要：细胞衰老是一个重要的生物学过程，是个体衰老的基础，并且是一种重要的抗癌机制。在细胞衰老的过程中，伴随着一系列的生理生化过程，基因的表达也发生着剧烈的变化，包括转录层面的调控以及转录后调控。而转录过程中，可变剪接（Alternative Splicing，AS）和选择性多聚腺苷酸化（Alternative Polyadenylation，APA）是重要的调控过程，并且都对细胞衰老产生一定的影响，且在细胞衰老过程中发生着显著的变化。随着高通量测序技术，尤其是第二代测序技术（Next Generation Sequencing，NGS）的发展，研究人员对全基因组APA和AS 进行深度测序和分析，发现两者具有一定程度的耦联关系，而在衰老细胞中并不明确。本课题以小鼠胚胎成纤文细胞（MEF细胞）为研究对象，利用三代测序方法（即由Pacific Biotechnology公司研发的ISO-seq技术，能够测出RNA的全长数据）测得MEF细胞总RNA全长数据，经生物信息学分析后，对取得的部分结果进行报告。30815
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毕业论文关键词：细胞衰老；可变剪接；选择性多聚腺苷酸化；三代测序（ISO-seq）
Research on the Coupling Relationship between Alternative Splicing and Alternative Polyadenylation in Cell Senescence
Abstract： Cell Senescence is an important biological process that is the basis of inpidual aging and is an important anti-cancer mechanism. In the process of cell senescence, accompanied by a series of physiological and biochemical processes, the expression of genes also undergoes dramatic changes, including transcriptional regulation and post-transcriptional regulation. Alternative Splicing (AS) and Alternative Polyadenylation (APA) are important regulatory processes in the process of transcription, they have important effect on Cell Senescence, and during the process of Cell Senescence significant changes have taken place on them. With the development of high throughput sequencing technology, especially the second generation sequencing (NGS), researchers carried out deep sequencing and analysis of whole genome APA and AS, found that the two had a certain degree of coupling relationship, but it’s not clear in the process of cell senescence. In this study, mouse embryonic fibroblasts (MEF) were used as the object of study. And we get the data of MEF full length transcripts by three-generation sequencing method (full-length cDNA sequencing method termed ISO-seq was established by Pacific Bioscience Company). Through the bioinformatics analysis, we report some of the results about the experiment in this article.
Key words: Cell Senescence；Alternative Splicing；Alternative Polyadenylation；ISO-seq
目 录
摘要4
关键词4
Abstract4
Key words4
引言（或绪论）5
1材料与方法 6
1.1研究对象 6
1.2实验材料 6
1.2.1 MEF细胞传代培养试剂 6
1.2.2 β-gal染色试剂 6
1.2.3 MEF细胞总RNA提取试剂 6
1.2.4 Qubit试剂 6
1.2.5 ISO-seq测序试剂 7
1.2.6 实验仪器 7
1.3实验方法 7
1.3.1 MEF细胞传代培养 7
1.3.2 衰老细胞的β-gal染色 7
1.3.3 MEF细胞总RNA提取 7
1.3.3.1 MEF细胞总RNA提取 7
1.3.3.2 RNAClean XP磁珠纯化 7
1.3.4 文库构建及上机测序 8
1.3.4.1 cDNA的合成 8
1.3.4.2 大规模PCR 8
1.3.4.3 用BluePinpin®系统进行片段选择 8
1.3.4.4 SMRTbellTM 文库构建及上机测序 9
1.4生物信息学处理 9
2结果与分析 10
2.1衰老细胞的β-gal染色结果 10
2.2 PCR循环数优化结果 10
2.3 Agilent 2100 Bioanalyzer进行RNA质检结果11
2.4测序所得数据情况 12
2.5 生物信息分析结果13
2.5.1对转录起始位点和poly（A）位点的统计13