title: Strand-Specific Transcriptome Sequencing Using SMART Technology.
abstract: Next-generation sequencing is empowering a deeper understanding of biology by enabling RNA expression analysis over the entire transcriptome with high sensitivity and a wide dynamic range. One powerful application within this field is stranded RNA sequencing (RNA-seq), which is necessary to distinguish overlapping genes and to conduct comprehensive annotation and quantification of long non-coding RNAs. Commonly used methods for generating strand-specific RNA-seq libraries are often complicated by protocols that require several rounds of enzymatic treatments and clean-up steps, making them time-intensive, insensitive, and unsuitable for processing several samples simultaneously. An additional challenge in the generation of RNA-seq libraries from total RNA involves the high amount of ribosomal RNA (rRNA) in the starting material. This unit presents streamlined workflows for generating strand-specific RNA-seq libraries from 10 ng to 1 µg total RNA, representing a minimum of 1000 cells, in less than 7 hr with minimal carryover rRNA. These methods allow scientists to evaluate the expression of all transcripts, including non-polyadenylated long non-coding RNAs, even in limited biological samples. Combination of the RNase H-based RiboGone rRNA removal system and SMARTer Stranded RNA-seq technology enables depletion of over 95% of rRNA from mammalian samples, and direct production of Illumina-ready libraries that maintain strand-of-origin information. An alternate method for low input of highly degraded samples is also presented. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View, California.
authors.forename: Magnolia
authors.initials: M
authors.lastname: Bostick
authors.affiliation: Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View, California.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bolduc
authors.affiliation: Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View, California.
authors.forename: Alisa
authors.initials: A
authors.lastname: Lehman
authors.affiliation: Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View, California.
authors.forename: Andrew
authors.initials: A
authors.lastname: Farmer
mesh_terms: D000818:Animals; D018076:DNA, Complementary / Q000235:genetics; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D062085:RNA, Long Noncoding / Q000235:genetics*; D012335:RNA, Ribosomal / Q000302:isolation & purification; D017423:Sequence Analysis, RNA / Q000379:methods*; D059467:Transcriptome
chemical_list: D018076:DNA, Complementary; D062085:RNA, Long Noncoding; D012335:RNA, Ribosomal
keywords: degraded RNA; non-coding RNA; rRNA removal; stranded RNA-seq