RNA-seq library construction for small quantity of cells with engineered transposase

您当前的位置：

首页 >

文章列表页 >

RNA-seq library construction for small quantity of cells with engineered transposase

Basic Medicine | 更新时间：2026-01-09

- RNA-seq library construction for small quantity of cells with engineered transposase
- Journal of Third Military Medical University Vol. 41, Issue 9, Pages: 866-870(2019)
- 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：
- 基金信息：
- DOI：10.16016/j.1000-5404.201812190
  CLC： Q55; Q782; Q785
- Received：25 December 2018，
  
  Revised：2019-01-31，
  
  Published：15 May 2019
- 稿件说明：
移动端阅览
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：

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

BUERMANS H P, DEN DUNNEN J T. Next generation sequencing technology: Advances and applications[J]. Biochim Biophys Acta, 2014, 1842(10): 1932-1941. DOI:10.1016/j.bbadis.2014.06.015.

WANG Z, GERSTEIN M, SNYDER M. RNA-Seq: a revolutionary tool for transcriptomics[J]. Nat Rev Genet, 2009, 10(1): 57-63. DOI:10.1038/nrg2484.

OZSOLAK F, MILOS P M. RNA sequencing: advances, challenges and opportunities[J]. Nat Rev Genet, 2011, 12(2): 87-98. DOI:10.1038/nrg2934.

RAMSKÖLD D, LUO S J, WANG Y C, et al . Full-length mRNA-Seq from single-cell levels of RNA and individual circulating tumor cells[J ] . Nat Biotechnol, 2012, 30(8): 777-782. DOI:10.1038/nbt.2282.

JAITIN D A, KENIGSBERG E, KEREN-SHAUL H, et al . Massively parallel single-cell RNA-seq for marker-free decomposition of tissues into cell types[J ] . Science, 2014, 343(6172): 776-779. DOI:10.1126/science.1247651.

PICELLI S, BJÖRKLUND Å K, FARIDANI O R, et al . Smart-seq2 for sensitive full-lengthtranscriptome profiling in single cells[J ] . Nat Methods, 2013, 10(11): 1096-1098. DOI:10.1038/nmeth.2639.

ZIEGENHAIN C, VIETH B, PAREKH S, et al . Comparative analysis of single-cell RNA sequencing methods[J ] .Mol Cell, 2017, 65(4): 631-643.e4. DOI:10.1016/j.molcel.2017.01.023.

ADEY A, MORRISON H G, ASAN, et al . Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition[J ] . Genome Biol, 2010, 11(12): R119. DOI:10.1186/gb-2010-11-12-r119..

CHEN C Y, XING D, TAN L Z, et al . Single-cell whole-genome analyses by linear amplification via transposon insertion (LIANTI)[J ] . Science, 2017, 356(6334): 189-194. DOI:10.1126/science.aak9787..

MAJOREK K A, DUNIN-HORKAWICZ S, STECZKIEWICZ K, et al . The RNase H-like superfamily: new members, comparative structural analysis and evolutionary classification[J ] . Nucleic Acids Res, 2014, 42(7): 4160-4179. DOI:10.1093/nar/gkt1414.

GERTZ J, VARLEY K E, DAVIS N S, et al .Transposase mediated construction of RNA-seq libraries[J ] . Genome Res, 2012, 22(1): 134-141. DOI:10.1101/gr.127373.111.

REZNIKOFF W S. Tn5 as a model for understanding DNA transposition[J]. Mol Microbiol, 2003, 47(5): 1199-1206. DOI:10.1046/j.1365-2958.2003.03382.x.

KIA A, GLOECKNER C, OSOTHPRAROP T, et al . Improved genome sequencing using an engineered transposase[J ] . BMC Biotechnology, 2017, 17(1): 6. DOI:10.1186/s12896-016-0326-1.

HENNIG B P, VEL TEN L, RACKE I, et al . Large-scale low-cost NGS library preparation using a robust Tn5 purification and tagmentation protocol[J ] . G3 (Bethesda), 2018, 8(1): 79-89. DOI:10.1534/g3.117.300257.

PICELLI S, BJÖRKLUND A K, REINIUS B, et al . Tn5 transposase and tagmentation procedures for massively scaled sequencing projects[J ] . Genome Res, 2014, 24(12): 2033-2040. DOI:10.1101/gr.177881.114.

0

Views

0

下载量

0

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Related Articles

Transcriptomic sequencing reveals that WNK2 promotes reactive oxygen species production in ovarian cancer cells

Analysis of transcriptomic characteristics of brain tissue in patients with Rasmussen encephalitis

Related Author

李凤杰

贾永琴

王延洲

杜秀蓉

曹兰琴

周丹

梁志清

Qiaoli LUO

Related Institution

Department of Microbiology, School of Basic Medical Sciences, Capital Medical University

Department of Neurosurgery, Sanbo Brain Hospital, Capital Medical University

⁰