Next Generation Sequencing Facilitates Quantitative Analysis of Col, myb30 and MYB30-OE Transcriptomes

total mRNA profiles of 4-day-old Col (WT), MYB30 knockout mutant (myb30) and MYB30-OE were generated by deep sequencing, in triplicate, Sequencing was carried out with the Illumina HiSeq 2000 platform and the resulting reads were mapped to the reference genome of Arabidopsis thaliana (TAIR10) with TopHat (http://tophat.cbcb.umd.edu). Transcript expression was evaluated by cuffdiff (http://cufflinks.cbcb.umd.edu), and transcript abundance was estimated by fragments per kilobase of exon model per million mapped fragments (FPKM). Differentially expressed genes were selected using Student’s t-test with P<0.05. qRT–PCR validation was performed using SYBR Green assays. mRNA profiles of 4-day old Col, myb30 and MYB30-OE were generated by deep sequencing, in triplicate, using Illumina HiSeq 2000.

本研究通过深度测序并设置三次生物学重复，获取了4日龄拟南芥哥伦比亚野生型（Col, Wild Type, WT）、MYB30基因敲除突变体（myb30）及MYB30过表达株系（MYB30-OE，OE为Overexpression缩写）的全mRNA转录组图谱。测序工作采用Illumina HiSeq 2000测序平台完成，所得测序读段通过TopHat工具（http://tophat.cbcb.umd.edu）比对至拟南芥（Arabidopsis thaliana）参考基因组TAIR10版本。转录本的表达水平通过cuffdiff软件（http://cufflinks.cbcb.umd.edu）进行评估，转录本丰度以每百万映射片段的外显子模型每千碱基片段数（Fragments Per Kilobase of exon model per Million mapped fragments, FPKM）进行估算。差异表达基因采用学生t检验进行筛选，筛选阈值设定为P<0.05。此外，采用SYBR Green染料法完成了qRT–PCR验证实验。本研究同样通过深度测序，采用Illumina HiSeq 2000平台并设置三次生物学重复，获取了4日龄Col、myb30及MYB30-OE的全mRNA转录组图谱。