Evaluation of a custom transcriptome sequencing library construction reagent with a small amount of cell input
10.3969/j.issn.1674-8115.2020.04.009
- VernacularTitle: 少量细胞输入的自制转录组测序文库构建试剂评测
- Author:
Lei DING
1
Author Information
1. Department of Immunology and Microbiology, Shanghai Jiao Tong University College of Basic Medical Sciences
- Publication Type:Journal Article
- Keywords:
A small amount of cell input;
Bioinformat-ics;
Next-generation sequencing;
Reagent comparison;
Transcriptome sequencing (RNA-seq)
- From:
Journal of Shanghai Jiaotong University(Medical Science)
2020;40(4):472-477
- CountryChina
- Language:Chinese
-
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 made by ourselves based on the SMART (switching mechanism at 5' end of RNA template) technology. Methods ¡¤ Four 8-week-old C57BL/6 female mice were randomly di-vided 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 from the peritoneal macrophages. cDNA library con-struction was performed with DIY reagent and TaKaRa reagent respectively. Finally, bioinformatics analysis was performed to compare the RNA sequencing results after use of different library construction reagents from different aspects, such as data quality, gene differential expression analysis, and KEGG (Kyoto encyclopedia of genes and genomes) pathway analysis. Results ¡¤ The results of bioinformatics analysis showed that the sample processed by the DIY reagent and TaKaRa reagent were both of good data quality, and the two reagents had comparative capabil-ity in transcripts capture. Gene coverage of the sequences both showed consistent uniformity. On top of these, the results of differential gene ex-pression analysis and gene pathway analysis were consistent. Conclusion ¡¤ Considering relatively great reduction in experimental cost for li-brary construction, the DIY reagent can replace expensive commercial reagent for library construction experiments with a small amount of cell input.
