纳米孔单分子测序

使用mRNA Dynabeads 纯化试剂盒进行poly(A)+ RNA的纯化。300 ng的poly(A)+ RNA使用T4连接酶和Nanopore RTA接头进行连接，使用SuperScriptsIII反转录酶对RNA进行反转录，形成RNA-DNA双链。产物使用1.8× (72 μl) VAHTS RNA纯化磁珠进行纯化，使用Qubit dsDNA HS array试剂盒进行定量，将RNA接头RMX连接至RNA:DNA双链的3' 末端RTA上。于是将75 μl的反应体系加载到GridION/MinION测序仪器的flow cell上。设置了两个重复，最终识别到22,953个基因的表达。

Poly(A)+ RNA was purified using the mRNA Dynabeads Purification Kit. 300 ng of the poly(A)+ RNA was ligated with the Nanopore RTA adapter using T4 ligase. Reverse transcription of the RNA was conducted using SuperScript III reverse transcriptase to generate RNA-DNA duplexes. The resulting products were purified with 1.8× (72 μL) VAHTS RNA Purification Magnetic Beads, and quantification was performed using the Qubit dsDNA HS Array Kit. The RNA adapter RMX was then ligated to the 3' terminal RTA of the RNA:DNA duplexes. Subsequently, 75 μL of the reaction mixture was loaded onto the flow cell of a GridION/MinION sequencing instrument. Two replicates were set up, and ultimately, the expression of 22,953 genes was detected.

搜集汇总

背景与挑战

背景概述

该数据集基于纳米孔单分子测序技术，通过poly(A)+ RNA纯化、反转录和测序流程，在林木次生生长的分子调控和环境胁迫机制项目中，识别了22,953个基因的表达。数据来源于国家重点研发计划项目，采用协议共享方式提供。