Chemical capping improves template switching and enhances sequencing of small RNAs.
Nucleic Acids Res
; 50(1): e2, 2022 01 11.
Article
em En
| MEDLINE
| ID: mdl-34581823
ABSTRACT
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Capuzes de RNA
/
Análise de Sequência de RNA
Limite:
Humans
Idioma:
En
Ano de publicação:
2022
Tipo de documento:
Article