title: Depletion of Ribosomal RNA Sequences from Single-Cell RNA-Sequencing Library.
abstract: Recent advances in single-cell RNA sequencing technologies have revealed high heterogeneity of gene expression profiles in individual cells. However, most current single-cell RNA-seq methods use oligo-dT priming in the reverse transcription steps and detect only polyA-positive for more accuracy, since there are also polyA-positive non-coding RNAs transcripts, not other important RNA species, such as polyA-negative noncoding RNA. Reverse transcription using random oligos enables detection of not only the noncoding RNA species without polyA tails, but also ribosomal RNA (rRNA). rRNA comprises more than 90% of the total RNA and should be depleted from the RNA-seq library to ensure efficient usage of the sequencing capacity. Commonly used hybridization-based rRNA depletion methods can preserve noncoding RNA in the standard RNA-seq library. However, such rRNA depletion methods require high input amounts of total RNA and do not work at the single-cell level or with limited input DNA. This unit describes a novel procedure for RNA-seq library construction from single cells or a minimal amount of RNA. A thermostable duplex-specific nuclease is used in this method to effectively remove ribosomal RNA sequences following whole-transcriptome amplification and sequencing library construction. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: QIAGEN GmbH, Hilden, Germany.
authors.forename: Nan
authors.initials: N
authors.lastname: Fang
authors.affiliation: QIAGEN GmbH, Hilden, Germany.
authors.forename: Rumeysa
authors.initials: R
authors.lastname: Akinci-Tolun
mesh_terms: D000818:Animals; D020869:Gene Expression Profiling / Q000379:methods; D015723:Gene Library; D006801:Humans; D012313:RNA / Q000235:genetics*; D012335:RNA, Ribosomal / Q000235:genetics / Q000302:isolation & purification*; D017423:Sequence Analysis, RNA / Q000379:methods*; D059010:Single-Cell Analysis / Q000379:methods; D059467:Transcriptome
chemical_list: D012335:RNA, Ribosomal; D012313:RNA
keywords: RNA-seq library construction; ribosomal RNA depletion; single cell RNA sequencing; whole transcriptome amplification