上海交通大学学报（医学版）

上海交通大学学报（医学版） ›› 2020, Vol. 40 ›› Issue (4): 472-.doi: 10.3969/j.issn.1674-8115.2020.04.009

• 论著·基础研究 • 上一篇    下一篇

少量细胞输入的自制转录组测序文库构建试剂评测

丁 蕾1, 2，高彩霞1, 2，刘兆远1, 2，陈 磊1, 2   

  1. 1. 上海交通大学基础医学院免疫学与微生物学系，上海 200025；2. 上海交通大学医学院，上海市免疫学研究所，上海 200025
  • 出版日期:2020-04-28 发布日期:2020-05-22
  • 作者简介:丁 蕾（1995—），女，硕士生；电子信箱：l.ding@sjtu.edu.cn。

Evaluation of a custom transcriptome sequencing library construction reagent with a small amount of cell input

DING Lei, GAO Cai-xia, LIU Zhao-yuan, CHEN Lei   

  1. 1. Department of Immunology and Microbiology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China; 2. Shanghai Institute of Immunology, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
  • Online:2020-04-28 Published:2020-05-22

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摘要： 目的·验证基于SMART（switching mechanism at 5 end of RNA template）技术自制的转录组测序文库构建试剂（DIY试剂）替代昂贵的商业化试剂TaKaRa SMART-Seq v4试剂盒（TaKaRa试剂）的可行性。方法·选取4只8周龄雌性C57BL/6小鼠，随机分为2组：一组作为对照组，不进行处理；另一组向小鼠腹腔内注射1 mL浓度为4%的巯基乙酸盐肉汤，诱导巨噬细胞。经过72 h后，分离腹腔巨噬细胞并提取RNA，用DIY试剂和TaKaRa试剂分别进行cDNA文库的构建。经二代测序后，利用生物信息学分析方法从数据质量、基因差异表达分析、KEGG（Kyoto encyclopedia of genes and genomes）通路富集分析3个方面探究不同建库试剂对转录组测序结果的影响。结果·DIY试剂和TaKaRa试剂处理的样品数据质量良好；2种试剂捕获转录本的能力相近；样品所测序列在基因上的覆盖度均匀且一致性较高；差异表达基因以及通路富集的分析结果基本一致。结论·DIY试剂可替代TaKaRa试剂进行少量细胞的转录组测序文库构建，并降低建库成本。

关键词: 少量细胞输入, 试剂比较, 二代测序, 转录组测序, 生物信息学

Abstract:

Objective · To verify the feasibility of replacing the expensive commercial reagent SMART-Seq v4 Ultra Low Input RNA Kit (hereinafter referred to as TaKaRa reagent) with a reagent (hereinafter referred to as DIY reagent) which was madeourselves based on the SMART (switching mechanism at 5 end of RNA template) technology. Methods · Four 8-week-old C57BL/6 female mice were randomly divided into two groups. One group did not receive any treatment as a control, and the other group was intraperitoneally injected with 1 mL of 4% thioglycollate broth to induce peritoneal macrophages. After 72 hours, RNA was extracted the peritoneal macrophages. cDNA library construction was performed with DIY reagent and TaKaRa reagent respectively. Finally, bioinformatics analysis was performed to compare the RNA sequencing results after of different library construction reagents different aspects, such as data quality, gene differential analysis, and KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis. Results · The results of bioinformatics analysis showed that the sample processedthe DIY reagent and TaKaRa reagent were both of good data quality, and the two reagents had comparative capability in transcripts capture. Gene coverage of the sequences both showed consistent uniformity. On top of these, the results of differential gene analysis and gene pathway analysis were consistent. Conclusion · Considering relatively great reduction in experimental cost for library construction, the DIY reagent can replace expensive commercial reagent for library construction experiments with a small amount of cell input.

Key words: a small amount of cell input, reagent comparison, next-generation sequencing, transcriptome sequencing (RNA-seq), bioinformatics