NEXTflex™ Small RNA Sequencing Kit v3
正常起始量的情况下,文库制备可完全免除凝胶纯化过程
更强的检测率,降低了测序成本
随机性接头降低连接偏差,获得更准确的原始样本量数据
文库制备起始量可低至1ng,此方案仍需进行凝胶纯化
AIR™ Ligase ,一种高效的截短型的T4 RNA连接酶,增加测序深度
可利用更大的样本量-10.5 µL的起始量样本
试剂盒中包含48种barcodes 运用于多重测序
经Illiumina 测序平台验证
随机性接头,减少小RNA 建库中的偏差,并可免除小RNA 建库中的凝胶纯化
NEXTflex™ Small RNA Sequencing Kit v3 使用的专利和正在申请中的专利技术为small RNA建库提供一个可以减少偏差的实验方案,可免除凝胶纯化或使用低样本起始量,这个方案适用于Illumina平台。Bioo Scientific 用以减少连接偏差的方法是在连接步骤中使用随机性接头,相比传统的方法而言,大大减少了偏差的产生。数据中偏差的减少,更加准确的反应出起始材料中small RNA的丰度。此外,还能够在更少的总reads数中检测到更多的miRNAs,提高small RNA的测序效率,降低测序成本。
NEXTflex Small RNA-Seq Kit v3 可免除Small RNA建库中的凝胶纯化过程,且降低样本起始量。试剂盒采用两种方法大幅降低接头二聚体的形成,以免除凝胶纯化。样品起始量为 ≥200 ng 的总RNA时,small RNA建库可通过减少接头二聚体的方式完全免除凝胶纯化过程。使用 NEXTflex Small RNA-Seq kit v3 进行免凝胶电泳建库可获得高miRNAs读取率。文库制备的样本起始量可少至1ng总RNA,需增加PCR循环数,获得无接头二聚体的文库(低样本起始量文库构建通常需要进行凝胶片段选择)。
Figure 1. 使用人类脑部总RNA两次重复建立small RNA文库,于Illumina Miseq平台测序。横坐标表示从不同文库中收集到的reads数目,纵坐标为检测到的iRNA组的平均数,测序数据中一组miRNA有≥20 reads数才定义为被检测到。组成曲线的点为检测到100组iRNA所需要的总reads数量。
低起始量的Small RNA 建库方案
减少接头二聚体的技术应用于small RNA建库试剂盒,也可降低文库制备时样本起始量。文库制备的样本起始量可少至5ng总RNA,增加PCR循环数,获得无接头二聚体的文库。 Fig.2 显示的是不同样品起始量时表达值的重复性
Figure 2. 使用 NEXTflex Small RNA-Seq kit v3,利用起始量为100 ng 和10 ng 人脑部总 RNA样本建库,并分析检测到的miRNA表达的差异。皮尔森相关系数如上显示。
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