Fig. 1

Single-molecule Nascent RNA Sequencing (FLEP-seq) captures various transient RNA intermediates produced during transcription termination. a Schematic of the FLEP-seq library preparation and sequencing. A 3′ adapter is ligated to nascent RNA for reverse transcription and full-length cDNA cloning. b Example of the RNA intermediates from gene DRM2. Upper panel: Nanopore reads are aligned to gene ordered by 3′ end position. The number of individual long reads (n) is indicated. Lower panel: coverage of Illumina short-read nascent RNA data from previous studies [39,40,41,42]. The gray dash line indicates the poly(A) site (pA). c Proportion of various RNA intermediates during termination. Readthrough transcripts (blue), 5′ cleavage products (red), 3′ cleavage products (yellow) and poly(A) transcripts (purple). d Sequencing summary for two biological replicates of FLEP-seq libraries in wildtype Col-0. e Advantage of Nanopore (upper) over Illumina [40] (lower) in separating reads from closely adjacent genes. The red dashed box highlights the intergenic region. f Meta-profile showing the 3′ end distribution of non-poly(A) + nascent RNA detected by the FLEP-seq, plaNET-seq [42] reads, and GRO-seq [40] near poly(A) site