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, researchers at Indiana University School of Medicine 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).
- Miller DF, Yan PS, Buechlein A, Rodriguez BA, Yilmaz AS, Goel S, Lin H, Collins-Burow B, Rhodes LV, Braun C, Pradeep S, Rupaimoole R, Dalkilic M, Sood AK, Burow ME, Tang H, Huang TH, Liu Y, Rusch DB, Nephew KP. (2013) A new method for stranded whole transcriptome RNA-seq. Methods [Epub ahead of print]. [abstact]