Intramolecular circularization increases efficiency of RNA sequencing and enables CLIP-Seq of nuclear RNA from human cells

RNA sequencing (RNA-Seq) is a powerful tool for analyzing the identity of cellular RNAs but is often limited by the amount of material available for analysis. In spite of extensive efforts employing existing protocols, we observed that it was not possible to obtain useful sequencing libraries from n...

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Bibliographic Details
Published in:Nucleic acids research 2015-06, Vol.43 (11), p.e75-e75
Main Authors: Chu, Yongjun, Wang, Tao, Dodd, David, Xie, Yang, Janowski, Bethany A, Corey, David R
Format: Article
Language:English
Subjects:
Argonaute Proteins - isolation & purification
Cell Line
Cell Nucleus - genetics
Computational Biology
Gene Library
Humans
Immunoprecipitation
Methods Online
RNA - chemistry
RNA - isolation & purification
RNA - radiation effects
RNA Ligase (ATP)
Sequence Analysis, RNA - methods
Templates, Genetic
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Summary:RNA sequencing (RNA-Seq) is a powerful tool for analyzing the identity of cellular RNAs but is often limited by the amount of material available for analysis. In spite of extensive efforts employing existing protocols, we observed that it was not possible to obtain useful sequencing libraries from nuclear RNA derived from cultured human cells after crosslinking and immunoprecipitation (CLIP). Here, we report a method for obtaining strand-specific small RNA libraries for RNA sequencing that requires picograms of RNA. We employ an intramolecular circularization step that increases the efficiency of library preparation and avoids the need for intermolecular ligations of adaptor sequences. Other key features include random priming for full-length cDNA synthesis and gel-free library purification. Using our method, we generated CLIP-Seq libraries from nuclear RNA that had been UV-crosslinked and immunoprecipitated with anti-Argonaute 2 (Ago2) antibody. Computational protocols were developed to enable analysis of raw sequencing data and we observe substantial differences between recognition by Ago2 of RNA species in the nucleus relative to the cytoplasm. This RNA self-circularization approach to RNA sequencing (RC-Seq) allows data to be obtained using small amounts of input RNA that cannot be sequenced by standard methods.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkv213