期刊
NATURE BIOTECHNOLOGY
卷 39, 期 8, 页码 978-+出版社
NATURE PORTFOLIO
DOI: 10.1038/s41587-021-00874-y
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资金
- National Natural Science Foundation of China [32070629]
- US National Science Foundation [MCB-1412379]
- National Institute of Environmental Health Sciences [ES002109]
- National Research Foundation of Singapore through the Singapore-MIT Alliance for Research and Technology Antimicrobial Resistance IRG
- MIT Toxicology Training grant [T32-ES007020]
- A*STAR, Singapore
- Swiss National Science Foundation
AQRNA-seq is a new method that allows accurate quantification of small RNAs in a sample, providing a quantitative map of the small RNA landscape in cells.
Current next-generation RNA-sequencing (RNA-seq) methods do not provide accurate quantification of small RNAs within a sample, due to sequence-dependent biases in capture, ligation and amplification during library preparation. We present a method, absolute quantification RNA-sequencing (AQRNA-seq), that minimizes biases and provides a direct, linear correlation between sequencing read count and copy number for all small RNAs in a sample. Library preparation and data processing were optimized and validated using a 963-member microRNA reference library, oligonucleotide standards of varying length, and RNA blots. Application of AQRNA-seq to a panel of human cancer cells revealed >800 detectable miRNAs that varied during cancer progression, while application to bacterial transfer RNA pools, with the challenges of secondary structure and abundant modifications, revealed 80-fold variation in tRNA isoacceptor levels, stress-induced site-specific tRNA fragmentation, quantitative modification maps, and evidence for stress-induced, tRNA-driven, codon-biased translation. AQRNA-seq thus provides a versatile means to quantitatively map the small RNA landscape in cells. AQRNA-seq allows accurate quantification of small RNAs.
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