FLEP-seq

FLEP-seq maps full-length nascent RNA transcripts to determine RNA polymerase II positioning, splicing status, polyadenylation site usage, and poly(A) tail length genome-wide in model organisms such as Arabidopsis.

Key Features:

• Simultaneous monitoring: Captures coordination between elongation, splicing, and polyadenylation on individual full-length transcripts, reporting RNA polymerase II positions and splicing status.
• Kinetics analysis: Calculates cotranscriptional splicing kinetics and detects polyadenylated transcripts retaining unspliced introns at defined posttranscriptional positions.
• Minimal RNA input: Library preparation requires as little as one nanogram of nascent RNA after removal of rRNA and tRNA.
• Sequencing platform compatibility: Supports long-read sequencing on Nanopore and PacBio platforms to capture full-length transcripts.
• Poly(A) analysis: Measures poly(A) tail length and identifies polyadenylation sites genome-wide.
• Comprehensive bioinformatic pipeline: Provides a pipeline for processing raw long-read sequencing data through downstream analyses.

Scientific Applications:

• Plant transcriptional regulation: Studying transcriptional regulation and RNA processing in plant models such as Arabidopsis.
• Cotranscriptional splicing studies: Investigating splicing dynamics and polymerase behavior at single-transcript resolution.
• Polyadenylation and tail dynamics: Characterizing polyadenylation site usage and poly(A) tail length effects on RNA fate.
• Transcriptional responses to stimuli: Examining how transcriptional dynamics influence cellular functions and responses to environmental stimuli.

Methodology:

A bioinformatic pipeline processes raw Nanopore or PacBio long-read sequencing data and performs downstream analyses including RNA polymerase II positioning, splicing status assessment, polyadenylation site identification, poly(A) tail length measurement, and calculation of cotranscriptional splicing kinetics.