title: A new method for stranded whole transcriptome RNA-seq.
abstract: This report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).
authors.affiliation: Medical Sciences, Indiana University School of Medicine, 1001 East 3rd St., Bloomington, IN 47405, United States. Electronic address: millerdf@indiana.edu.
authors.forename: David F B
authors.initials: DF
authors.lastname: Miller
authors.forename: Pearlly S
authors.initials: PS
authors.lastname: Yan
authors.forename: Aaron
authors.initials: A
authors.lastname: Buechlein
authors.forename: Benjamin A
authors.initials: BA
authors.lastname: Rodriguez
authors.forename: Ayse S
authors.initials: AS
authors.lastname: Yilmaz
authors.forename: Shokhi
authors.initials: S
authors.lastname: Goel
authors.forename: Hai
authors.initials: H
authors.lastname: Lin
authors.forename: Bridgette
authors.initials: B
authors.lastname: Collins-Burow
authors.forename: Lyndsay V
authors.initials: LV
authors.lastname: Rhodes
authors.forename: Chris
authors.initials: C
authors.lastname: Braun
authors.forename: Sunila
authors.initials: S
authors.lastname: Pradeep
authors.forename: Rajesha
authors.initials: R
authors.lastname: Rupaimoole
authors.forename: Mehmet
authors.initials: M
authors.lastname: Dalkilic
authors.forename: Anil K
authors.initials: AK
authors.lastname: Sood
authors.forename: Matthew E
authors.initials: ME
authors.lastname: Burow
authors.forename: Haixu
authors.initials: H
authors.lastname: Tang
authors.forename: Tim H
authors.initials: TH
authors.lastname: Huang
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Liu
authors.forename: Douglas B
authors.initials: DB
authors.lastname: Rusch
authors.forename: Kenneth P
authors.initials: KP
authors.lastname: Nephew
mesh_terms: D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D058977:Molecular Sequence Annotation; D020641:Polymorphism, Single Nucleotide; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012335:RNA, Ribosomal / Q000737:chemistry / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000737:chemistry; D017423:Sequence Analysis, RNA
chemical_list: D020033:Protein Isoforms; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D012260:Ribonucleases
keywords: DSN; Duplex-specific nuclease; FLgRNA; Gene expression; LgRNA; RNA-seq; Transcriptome; duplex-specific nuclease; fragmented LgRNA; large total RNA >200 nt; smRNA; small total RNA <200 nt
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