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

  • Yasunori IchihashiEmail author
  • Atsushi Fukushima
  • Arisa Shibata
  • Ken Shirasu
Part of the Methods in Molecular Biology book series (MIMB, volume 1830)


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.

Key words

Bioinformatics Breath adapter directional sequencing Differential regulatory analysis Network analysis RNA-seq Transcriptome 



AMPure XP bead resuspension buffer


Breath adapter directional sequencing


Differentially expressed genes


Digital gene expression


Differential regulatory analysis


Lysis/binding buffer


Low-salt buffer


Next generation sequencing




Washing buffer A


Washing buffer B



This work was supported by PRESTO, Japan Science and Technology Agency (JPMJPR15Q2) to YI and JSPS KAKENHI Grant Number 17 K07663 to AF, 15H05959 and 17H06172 to KS.

Supplementary material

435160_1_En_11_MOESM1_ESM.xlsx (40 kb)
Supplementary File 1: (XLSX 40 kb)


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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Yasunori Ichihashi
    • 1
    • 2
    • 3
    Email author
  • Atsushi Fukushima
    • 1
  • Arisa Shibata
    • 1
  • Ken Shirasu
    • 1
    • 4
  1. 1.RIKEN Center for Sustainable Resource ScienceYokohamaJapan
  2. 2.JST, PRESTOKawaguchiJapan
  3. 3.RIKEN BioResource Research CenterTsukubaJapan
  4. 4.Graduate School of ScienceThe University of TokyoTokyoJapan

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