【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Ta KaRa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。更多还原

【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 C57 BL/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 from 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 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 capability in transcripts capture. Gene coverage of the sequences both showed consistent uniformity. On top of these, the results of differential gene expression 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.更多还原