Journal
NUCLEIC ACIDS RESEARCH
Volume 50, Issue 1, Pages -Publisher
OXFORD UNIV PRESS
DOI: 10.1093/nar/gkab861
Keywords
-
Categories
Funding
- New England Biolabs, Inc.
Ask authors/readers for more resources
Template-switching reverse transcription using CapTS-seq, which combines chemical capping and template switching, shows improved efficiency and reduced bias in sequencing small RNAs. The study provides evidence that the 5' cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition, crucial for template switching efficiency. Deploying CapTS-seq for sequencing small RNAs consistently improves library quality compared to traditional methods.
Template-switching reverse transcription is widely used in RNA sequencing for low-input and low-quality samples, including RNA from single cells or formalin-fixed paraffin-embedded (FFPE) tissues. Previously, we identified the native eukaryotic mRNA 5' cap as a key structural element for enhancing template switching efficiency. Here, we introduce CapTS-seq, a new strategy for sequencing small RNAs that combines chemical capping and template switching. We probed a variety of non-native synthetic cap structures and found that an unmethylated guanosine triphosphate cap led to the lowest bias and highest efficiency for template switching. Through cross-examination of different nucleotides at the cap position, our data provided unequivocal evidence that the 5' cap acts as a template for the first nucleotide in reverse transcriptase-mediated post-templated addition to the emerging cDNA-a key feature to propel template switching. We deployed CapTS-seq for sequencing synthetic miRNAs, human total brain and liver FFPE RNA, and demonstrated that it consistently improves library quality for miRNAs in comparison with a gold standard template switching-based small RNA-seq kit.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available