Single-molecule targeted accessibility and methylation sequencing of centromeres, telomeres and rDNAs in Arabidopsis.

The short read-length of next-generation sequencing makes it challenging to characterize highly repetitive regions (HRRs) such as centromeres, telomeres and ribosomal DNAs. Based on recent strategies that combined long-read sequencing and exogenous enzymatic labelling of open chromatin, we developed single-molecule targeted accessibility and methylation sequencing (STAM-seq) in plants by further integrating nanopore adaptive sampling to investigate the HRRs in wild-type Arabidopsis and DNA methylation mutants that are defective in CG- or non-CG methylation. We found that CEN180 repeats show higher chromatin accessibility and lower DNA methylation on their forward strand, individual rDNA units show a negative correlation between their DNA methylation and accessibility, and both accessibility and CHH methylation levels are lower at telomere compared to adjacent subtelomeric region. Moreover, DNA methylation-deficient mutants showed increased chromatin accessibility at HRRs, consistent with the role of DNA methylation in maintaining heterochromatic status in plants. STAM-seq can be applied to study accessibility and methylation of repetitive sequences across diverse plant species.

An atlas of plant full-length RNA reveals tissue-specific and monocots-dicots conserved regulation of poly(A) tail length.

Poly(A) tail is a hallmark of eukaryotic messenger RNA and its length plays an essential role in regulating mRNA metabolism. However, a comprehensive resource for plant poly(A) tail length has yet to be established. Here, we applied a poly(A)-enrichment-free, nanopore-based method to profile full-length RNA with poly(A) tail information in plants. Our atlas contains over 120 million polyadenylated mRNA molecules from seven different tissues of Arabidopsis, as well as the shoot tissue of maize, soybean and rice. In most tissues, the size of plant poly(A) tails shows peaks at approximately 20 and 45 nucleotides, while the poly(A) tails in pollen exhibit a distinct pattern with strong peaks centred at 55 and 80 nucleotides. Moreover, poly(A) tail length is regulated in a gene-specific manner-mRNAs with short half-lives in general have long poly(A) tails, while mRNAs with long half-lives are featured with relatively short poly(A) tails that peak at ~45 nucleotides. Across species, poly(A) tails in the nucleus are almost twice as long as in the cytoplasm. Our comprehensive dataset lays the groundwork for future functional and evolutionary studies on poly(A) tail length regulation in plants.

Landscape of transcription termination in Arabidopsis revealed by single-molecule nascent RNA sequencing.

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.

HITAC-seq enables high-throughput cost-effective sequencing of plasmids and DNA fragments with identity.

DNA sequencing is vital for many aspects of biological research and diagnostics. Despite the development of second and third generation sequencing technologies, Sanger sequencing has long been the only choice when required to precisely track each sequenced plasmids or DNA fragments. Here, we report a complete set of novel barcoding and assembling system, Highly-parallel Indexed Tagmentation-reads Assembled Consensus sequencing (HITAC-seq), that could massively sequence and track the identities of each individual sequencing sample. With the cost of much less than that of single read of Sanger sequencing, HITAC-seq can generate high-quality contiguous sequences of up to 10 kilobases or longer. The capability of HITAC-seq was confirmed through large-scale sequencing of thousands of plasmid clones and hundreds of amplicon fragments using approximately 100 pg of input DNAs. Due to its long synthetic length, HITAC-seq was effective in detecting relatively large structural variations, as demonstrated by the identification of a ∼1.3 kb Copia retrotransposon insertion in the upstream of a likely maize domestication gene. Besides being a practical alternative to traditional Sanger sequencing, HITAC-seq is suitable for many high-throughput sequencing and genotyping applications.

FLEP-seq: simultaneous detection of RNA polymerase II position, splicing status, polyadenylation site and poly(A) tail length at genome-wide scale by single-molecule nascent RNA sequencing.

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.

FlsnRNA-seq: protoplasting-free full-length single-nucleus RNA profiling in plants.

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.

Structural characterization and immunomodulatory activity of a water-soluble polysaccharide from Ganoderma leucocontextum fruiting bodies.

Ganoderma leucocontextum is a new species of Ganoderma discovered in 2014. Up to now, the structural characteristics and immunoregulatory activity of its polysaccharides remain virtually unknown. In this study, a water-soluble polysaccharide termed, GLP-3, was purified from G. leucocontextum by ultrafiltration and column chromatography. The results revealed that GLP-3 mainly consisted of glucose (92.7 %) and its weight average molecular weight was 159.7 kDa. The structural analysis indicated that the backbone of GLP-3 was →4)-α-D-Glcp-(1→4,6)-ß-D-Glcp-(1→ with a ß-Glcp-(1→ branch. Atomic force microscopy and Congo red experiments revealed that GLP-3 might possess a globular structure with triple-helix conformation in water. Moreover, GLP-3 was recognized by toll-like receptor 2 (TLR2) and exerted immunomodulatory effects via activating mitogen-activated protein kinases (MAPKs), phosphatidylinositol-3-kinase (PI3K)/Akt and nuclear factor-κB (NF-κB) signaling pathways in RAW 264.7 macrophages. Collectively, these results suggested that GLP-3 could be developed as a potential functional food ingredient for immunomodulation.