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基于工程化转座酶的少量细胞RNA-seq文库构建的研究
RNA-seq library construction for small quantity of cells with engineered transposase
- 第三军医大学学报 2019年41卷第9期 页码：866-870
- 作者机构：
1.400038 重庆，陆军军医大学(第三军医大学)基础医学院生物医学分析测试中心
2.400715 重庆，西南大学生命科学院分子发育生物学实验室
- 作者简介：
[万瑛，E-mail:wanying516@foxmail.comWAN Ying, E-mail:wanying516@foxmail.com
- 基金信息：
DOI：10.16016/j.1000-5404.201812190
中图分类号： Q55; Q782; Q785收稿：2018-12-25，
修回：2019-01-31，
纸质出版：2019-05-15
- 稿件说明：
移动端阅览
许昊, 刘小亮, 胡玲, 等. 基于工程化转座酶的少量细胞RNA-seq文库构建的研究[J]. 第三军医大学学报, 2019,41(9):866-870.
Hao XU, Xiaoliang LIU, Ling HU, et al. RNA-seq library construction for small quantity of cells with engineered transposase[J]. Journal of Third Military Medical University, 2019, 41(9): 866-870.
许昊, 刘小亮, 胡玲, 等. 基于工程化转座酶的少量细胞RNA-seq文库构建的研究[J]. 第三军医大学学报, 2019,41(9):866-870. DOI： 10.16016/j.1000-5404.201812190.
Hao XU, Xiaoliang LIU, Ling HU, et al. RNA-seq library construction for small quantity of cells with engineered transposase[J]. Journal of Third Military Medical University, 2019, 41(9): 866-870. DOI： 10.16016/j.1000-5404.201812190.
摘要
目的
2
优化Tn5转座酶纯化和组装过程，建立少量细胞高质量转录组深度测序文库(RNA-Seq)的构建策略。
方法
2
构建Tn5转座酶表达载体pSUMO-Tn5，优化Tn5转座酶表达、亲和纯化和组装过程，比较不同组装方式的Tn5转座酶构建少量RAW264.7细胞RNA-seq文库效率。
结果
2
pSUMO-Tn5转化细菌高效表达可溶性Tn5转座酶，亲和纯化获得高纯度Tn5蛋白，超滤纯化的Tn5转座体显著降低少量细胞RNA-Seq中的无效数据(
P
<
0.05)。
结论
2
成功表达和纯化Tn5转座酶，超滤后Tn5转座体可降低少量细胞RNA-seq文库构建中的非特异扩增，建立一种高效的少量细胞RNA-Seq策略。
Abstract
Objective
2
To build a construction strategy for RNA-seq library for small quantity of cells by optimizing Tn5 transposase assembly and purification workflow.
Methods
2
Recombinant plasmid pSUMO-Tn5 was constructed by homologous recombination. After Tn5 protein was induced to express
the product was purified with affinity chromatography followed by optimization of Tn5 assembly. Then we constructed RNA-seq library using RAW264.7 cells and analyzed the number of genes detected and the ratio of unmapped reads.
Results
2
Soluble Tn5 protein was expressed in Codon Plus BL21 with the recombinant plasmid pSUMO-Tn5. Tn5 with high purity was obtained through affinity purification. RNA-seq library for small quantity of RAW264.7 cells was successfully constructed. After the optimization of Tn5 assembly
nonspecific amplification was remarkably reduced compared with the control (
P
<
0.05).
Conclusion
2
Tn5 protein is effectively expressed and purified. Ultra-filtered Tn5 transposase can reduce the nonspecific amplification in RNA-seq
providing a new strategy of RNA-seq for small quantity of cells.
关键词
Keywords
references
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