[1]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(09):866-870.
 XU Hao,LIU Xiaoliang,HU Ling,et al.RNA-seq library construction for small quantity of cells with engineered transposase[J].J Third Mil Med Univ,2019,41(09):866-870.
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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(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
41卷
期数:
2019年第09期
页码:
866-870
栏目:
基础医学
出版日期:
2019-05-15

文章信息/Info

Title:
RNA-seq library construction for small quantity of cells with engineered transposase
作者:
许昊刘小亮胡玲于海礼黄毅李洪涛万瑛
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;西南大学生命科学院分子发育生物学实验室
Author(s):
XU Hao LIU Xiaoliang HU Ling YU Haili HUANG Yi LI Hongtao WAN Ying

Biomedical Analysis Center, College of Basic Medical Sciences, Army Medical University (Third Military Medical University), Chongqing, 400038; Laboratory of Molecular Developmental Biology, School of Life Sciences, Southwest University, Chongqing 400715, China

关键词:
Tn5转座酶RNA-seq文库构建
Keywords:
Tn5 transposase RNA-seq library construction
分类号:
Q55;Q782;Q785
文献标志码:
A
摘要:

目的优化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:

ObjectiveTo build a construction strategy for RNAseq library for small quantity of cells by optimizing Tn5 transposase assembly and purification workflow. MethodsRecombinant 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. ResultsSoluble 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). ConclusionTn5 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.

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更新日期/Last Update: 2019-05-08