BrAD-seq: Breath Adapter Directional sequencing: a streamlined, ultra-simple and fast library preparation protocol for strand specific mRNA library construction

Abstract

Next Generation Sequencing (NGS) is driving rapid advancement in biological understanding and RNA-sequencing (RNA-seq) has become an indispensable tool for biology and medicine. There is a growing need for access to these technologies although preparation of NGS libraries remains a bottleneck to wider adoption. Here we report a novel method for the production of strand specific RNA-seq libraries utilizing the terminal breathing of double-stranded cDNA to capture and incorporate a sequencing adapter. Breath Adapter Directional sequencing (BrAD-seq) reduces sample handling and requires far fewer enzymatic steps than most available methods to produce high quality strand-specific RNA-seq libraries. The method we present is optimized for 3-prime Digital Gene Expression (DGE) libraries and can easily extend to full transcript coverage shotgun (SHO) type strand-specific libraries and is modularized to accommodate a diversity of RNA and DNA input materials. BrAD-seq offers a highly streamlined and inexpensive option for RNA-seq libraries.

Abscisic acid‐dependent histone demethylation during postgermination growth arrest in Arabidopsis

“…Plants of the same age were used as mock controls. Libraries for RNA-seq were prepared by the Breath Adapter Directional sequencing method (Townsley, Covington, Ichihashi, Zumstein, & Sinha, 2015). The libraries were sequenced by Next-Seq 500 (Illumina).…”

Nuclear Transcriptomes at High Resolution Using Retooled INTACT

“…Nuclei were isolated using p35S:NTF2 from four biological replicate samples of 200 root tips and used to generate nuclear RNA-seq libraries by rRNA subtraction and random-primer-enabled cDNA synthesis (Townsley et al, 2015). Total RNA-seq libraries were constructed with oligo dT-selected RNA.…”

“…Total and Nuclear RNA Extraction, rRNA Degradation, and Short-Read Library Preparation Total poly(A) + RNA was extracted from frozen tissue using polysome extraction buffer (Mustroph et al, 2009), followed by direct capture of poly(A) + RNA using a biotinylated oligo dT according to Townsley et al (2015). Nuclear RNA was extracted from 66,000 to 117,000 (Supplemental Table S2A) INTACT-purified nuclei using the RNeasy Micro kit (Qiagen), DNase-treated with Turbo DNase I (Thermo Fisher Scientific) and processed with Agencourt RNAClean XP beads (Beckman Coulter), per the manufacturer's' instructions.…”

“…For a full bench protocol, see Supplemental Appendix II. RNA-seq libraries of pre/rRNA subtracted nuclear and poly(A) + selected total mRNA were prepared as conventional non-strand specific libraries described by Townsley et al (2015).…”

Light-induced indeterminacy alters shade avoiding tomato leaf morphology

“…Heteroblasty RNA-Seq was performed using BrAD-seq, a strand-specific digital gene expression method (Townsley et al, 2015). As briefly as possible, tissues were processed and lysed as described by Kumar et al (2012) with the following modifications: (1) wash volumes of Washing Buffer A, Washing Buffer B, and Low Salt Buffer were 300 mL each, and buffers were chilled on ice prior to use; and (2) mRNA elution was done into 16 mL of 10 mM Tris-HCl, pH 8, containing 1 mM b-mercaptoethanol.…”