›› 2020, Vol. 40 ›› Issue (4): 472-.doi: 10.3969/j.issn.1674-8115.2020.04.009

• Original article (Basic research) • Previous Articles     Next Articles

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

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