High Impact Gene Discovery: Simple Strand-Specific mRNA Library Construction and Differential Regulatory Analysis Based on Gene Co-Expression Network.

Plant transcription factors have potential to behave as hubs in gene regulatory networks through altering the expression of many downstream genes, and identification of such hub transcription factors strongly enhances our understating of biological processes. Transcriptome analysis has become a staple of gene expression analyses. In addition to current advances in Next Generation Sequencing (NGS) technology, various methods for mRNA library construction and downstream data analyses have been enthusiastically developed. Here, we describe Breath Adapter Directional sequencing (BrAD-seq), a simple strand-specific mRNA library preparation for the Illumina platform, allowing easy scaling of transcriptome experiments with low reagent and labor costs. This protocol includes our recent modifications and the detailed practical procedure for BrAD-seq. Because extracting biological meanings from large-scale transcriptome data presents a significant challenge, we also describe a new analytical method that goes beyond differential expression. Differential regulatory analysis (DRA) is based on a gene co-expression network to address which regulatory factor or factors have the ability to predict the abundance of differentially expressed genes between two groups or conditions. This protocol provides a ready-to-use informatics pipeline from raw sequence data to DRA for plant transcriptome datasets.