Title
An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments
Academic Program
Biochemistry & Molecular Pharmacology
UMMS Affiliation
Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Publication Date
2015-1
Document Type
Article
Disciplines
Biochemistry | Bioinformatics | Computational Biology | Genetics and Genomics | Nucleic Acids, Nucleotides, and Nucleosides
Abstract
Deep sequencing of strand-specific cDNA libraries is now a ubiquitous tool for identifying and quantifying RNAs in diverse sample types. The accuracy of conclusions drawn from these analyses depends on precise and quantitative conversion of the RNA sample into a DNA library suitable for sequencing. Here, we describe an optimized method of preparing strand-specific RNA deep sequencing libraries from small RNAs and variably sized RNA fragments obtained from ribonucleoprotein particle footprinting experiments or fragmentation of long RNAs. Our approach works across a wide range of input amounts (400 pg to 200 ng), is easy to follow and produces a library in 2-3 days at relatively low reagent cost, all while giving the user complete control over every step. Because all enzymatic reactions were optimized and driven to apparent completion, sequence diversity and species abundance in the input sample are well preserved.
Rights and Permissions
Copyright The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
DOI of Published Version
10.1093/nar/gku1235
Source
Nucleic Acids Res. 2015 Jan;43(1):e2. doi: 10.1093/nar/gku1235. Epub 2014 Dec 12. Link to article on publisher's site
Journal/Book/Conference Title
Nucleic acids research
Related Resources
PubMed ID
25505164
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License
Repository Citation
Heyer EE, Ozadam H, Ricci EP, Cenik C, Moore MJ. (2015). An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments. GSBS Student Publications. https://doi.org/10.1093/nar/gku1235. Retrieved from https://escholarship.umassmed.edu/gsbs_sp/1890
Included in
Biochemistry Commons, Bioinformatics Commons, Computational Biology Commons, Nucleic Acids, Nucleotides, and Nucleosides Commons