title: Next-Generation Sequencing RNA-Seq Library Construction.
abstract: This unit presents protocols for construction of next-generation sequencing (NGS) directional RNA sequencing libraries for the Illumina HiSeq and MiSeq from a wide variety of input RNA sources. The protocols are based on the New England Biolabs (NEB) small RNA library preparation set for Illumina, although similar kits exist from different vendors. The protocol preserves the orientation of the original RNA in the final sequencing library, enabling strand-specific analysis of the resulting data. These libraries have been used for differential gene expression analysis and small RNA discovery and are currently being tested for de novo transcriptome assembly. The protocol is robust and applicable to a broad range of RNA input types and RNA quality, making it ideal for high-throughput laboratories.
authors.affiliation: Genomic Sequencing and Analysis Facility, University of Texas at Austin, Austin, Texas.
authors.forename: Jessica
authors.initials: J
authors.lastname: Podnar
authors.affiliation: Genomic Sequencing and Analysis Facility, University of Texas at Austin, Austin, Texas.
authors.forename: Heather
authors.initials: H
authors.lastname: Deiderick
authors.affiliation: Genomic Sequencing and Analysis Facility, University of Texas at Austin, Austin, Texas.
authors.forename: Gabriella
authors.initials: G
authors.lastname: Huerta
authors.affiliation: Genomic Sequencing and Analysis Facility, University of Texas at Austin, Austin, Texas.
authors.forename: Scott
authors.initials: S
authors.lastname: Hunicke-Smith
mesh_terms: D000818:Animals; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D012313:RNA / Q000235:genetics*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D012313:RNA
keywords: NGS; RNA-Seq; gene expression; library construction; strand-specific; transcriptome