The broad application of single-cell RNA profiling in plants has been hindered by the prerequisite of protoplasting that requires digesting the cell walls from different types of plant tissues. Here, we present a protoplasting-free approach, flsnRNA-seq, for large-scale full-length RNA profiling at a single-nucleus level in plants using isolated nuclei. Combined with 10x Genomics and Nanopore long-read sequencing, we validate the robustness of this approach in Arabidopsis root cells and the developing endosperm. Sequencing results demonstrate that it allows for uncovering alternative splicing and polyadenylation-related RNA isoform information at the single-cell level, which facilitates characterizing cell identities.

Background The dynamic process of transcription termination produces transient RNA intermediates that are difficult to distinguish from each other via short-read sequencing methods. Results Here, we use single-molecule nascent RNA sequencing to characterize the various forms of transient RNAs during termination at genome-wide scale in wildtype Arabidopsis and in atxrn3, fpa, and met1 mutants. Our data reveal a wide range of termination windows among genes, ranging from ~ 50 nt to over 1000 nt. We also observe efficient termination before downstream tRNA genes, suggesting that chromatin structure around the promoter region of tRNA genes may block pol II elongation. 5′ Cleaved readthrough transcription in atxrn3 with delayed termination can run into downstream genes to produce normally spliced and polyadenylated mRNAs in the absence of their own transcription initiation. Consistent with previous reports, we also observe long chimeric transcripts with cryptic splicing in fpa mutant; but loss of CG DNA methylation has no obvious impact on termination in the met1 mutant. Conclusions Our method is applicable to establish a comprehensive termination landscape in a broad range of species.

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Elongation, splicing and polyadenylation are fundamental steps of transcription, and studying their coordination requires simultaneous monitoring of these dynamic processes on one transcript. We recently developed a full-length nascent RNA sequencing method in the model plant Arabidopsis that simultaneously detects RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale. This method allows calculation of the kinetics of cotranscriptional splicing and detects polyadenylated transcripts with unspliced introns retained at specific positions posttranscriptionally. Here we describe a detailed protocol for this method called FLEP-seq (full-length elongating and polyadenylated RNA sequencing) that is applicable to plants. Library production requires as little as one nanogram of nascent RNA (after rRNA/tRNA removal), and either Nanopore or PacBio platforms can be used for sequencing. We also provide a complete bioinformatic pipeline from raw data processing to downstream analysis. The minimum time required for FLEP-seq, including RNA extraction and library preparation, is 36 h. The subsequent long-read sequencing and initial data analysis ranges between 31 and 40 h, depending on the sequencing platform.

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