cP-RNA-seq for tRNA half sequencing.

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Standard RNA sequencing (RNA-seq) has limitations in capturing short non-coding RNAs (sncRNAs) with non-standard terminal structures.
• Many abundant sncRNAs, such as tRNA halves, possess 2',3'-cyclic phosphates (cP) incompatible with standard RNA-seq protocols.
• This incompatibility hinders comprehensive analysis of sncRNA repertoires and their biological roles.

Purpose of the Study:

• To introduce and detail an updated protocol for cP-RNA-seq, a method designed for sequencing sncRNAs with cyclic phosphates.
• To enable the global identification and characterization of tRNA halves and other cP-containing sncRNAs.
• To overcome the limitations of standard RNA-seq for analyzing specific sncRNA classes.

Main Methods:

• Developed cP-RNA-seq, a method that selectively amplifies and sequences sncRNAs containing a 2',3'-cyclic phosphate (cP) at their 3'-end.
• The protocol involves phosphatase treatment followed by periodate treatment to cleave non-cP containing sncRNAs.
• Adaptor ligation and cDNA amplification are then exclusively performed on cP-containing sncRNAs.

Main Results:

• cP-RNA-seq successfully enriches and sequences tRNA halves and other sncRNAs with 3'-cP termini.
• The method demonstrates broad applicability across various transcriptomes for comprehensive sncRNA profiling.
• The updated protocol utilizes only commercially available reagents.

Conclusions:

• cP-RNA-seq provides a robust and accessible solution for the global identification of tRNA halves and other cyclic phosphate-containing sncRNAs.
• This method significantly enhances the scope of sncRNA analysis beyond the capabilities of standard RNA-seq.
• The protocol facilitates deeper understanding of the diverse roles of sncRNAs in biological processes.