Biochemistry & Molecular Pharmacology
Department of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Medical Subject Headings
DNA, Circular; DNA, Single-Stranded; Electrophoresis, Polyacrylamide Gel; *Gene Library; High-Throughput Nucleotide Sequencing; MicroRNAs; Reverse Transcriptase Polymerase Chain Reaction; Sequence Analysis, RNA
Biochemistry | Bioinformatics | Computational Biology | Genetics and Genomics | Nucleic Acids, Nucleotides, and Nucleosides
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.
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Citation: Nucleic Acids Res. 2015 Jan;43(1):e2. doi: 10.1093/nar/gku1235. Epub 2014 Dec 12. Link to article on publisher's site
Nucleic acids research
Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License
Heyer, Erin E.; Ozadam, Hakan; Ricci, Emiliano P.; Cenik, Can; and Moore, Melissa J., "An optimized kit-free method for making strand-specific deep sequencing libraries from RNA fragments" (2015). GSBS Student Publications. 1890.