title: Intramolecular circularization increases efficiency of RNA sequencing and enables CLIP-Seq of nuclear RNA from human cells.
abstract: 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.
authors: Departments of Pharmacology and Biochemistry, 6001 Forest Park Road, Dallas, TX 75390-9041, USA.
authors: Yongjun
authors: Y
authors: Chu
authors: Quantitative Biomedical Research Center, Department of Clinical Science, 6001 Forest Park Road, Dallas, TX 75390-9041, USA.
authors: Tao
authors: T
authors: Wang
authors: Departments of Pharmacology and Biochemistry, 6001 Forest Park Road, Dallas, TX 75390-9041, USA.
authors: David
authors: D
authors: Dodd
authors: Quantitative Biomedical Research Center, Department of Clinical Science, 6001 Forest Park Road, Dallas, TX 75390-9041, USA Simmons Cancer Center, UT Southwestern Medical Center, 6001 Forest Park Road, Dallas, TX 75390-9041, USA.
authors: Yang
authors: Y
authors: Xie
authors: Departments of Pharmacology and Biochemistry, 6001 Forest Park Road, Dallas, TX 75390-9041, USA.
authors: Bethany A
authors: BA
authors: Janowski
authors: Departments of Pharmacology and Biochemistry, 6001 Forest Park Road, Dallas, TX 75390-9041, USA david.corey@utsouthwestern.edu.
authors: David R
authors: DR
authors: Corey
mesh_terms: D060565:Argonaute Proteins / Q000302:isolation & purification; D002460:Cell Line; D002467:Cell Nucleus / Q000235:genetics*; D019295:Computational Biology; D015723:Gene Library; D006801:Humans; D047468:Immunoprecipitation; D012313:RNA / Q000737:chemistry* / Q000302:isolation & purification / Q000528:radiation effects; D011130:RNA Ligase (ATP); D000079962:RNA, Circular; D017423:Sequence Analysis, RNA / Q000379:methods*; D013698:Templates, Genetic
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D000079962:RNA, Circular; D012313:RNA; D011130:RNA Ligase (ATP)