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.
Soybean DCL2 favors long inverted repeat-derived double-stranded RNA as its substrate, and generates primary 22-nt siRNAs to regulate seed coat color and silence transposable elements.
Fabrication strategies for programmed hydrogels that provide precise spatial control with predetermined responses to external stimuli are highly desirable. In this study, a partially reversible light‐driven assembly (PRLDA) method is introduced to construct multiresponsive hydrogels utilizing microgel (MG) particle building blocks (swollen diameter of 107 nm). No other material is required to prepare the gels beyond the MGs themselves. Facile preparation of multiresponsive hydrogels that are reversibly responsive to light, pH, and temperature using phototriggered covalent interlinking of coumarin‐based MGs is demonstrated. The gels have phototuneable moduli and swelling ratios and show light‐assisted healing and reshaping. Remarkably, the intrinsic fluorescence of the gels undergoes a reversible light‐triggered wavelength‐shift. The emission peak blueshifted from 420 to 390 nm upon irradiation with 365 nm light. The PRLDA gels can be constructed using either positive or negative photopatterning. It is shown that the gels can be exploited for multiresponsive cytocompatible actuators, grippers, and ON/OFF circuit components as well as anticounterfeit gels. The PRLDA method provides new insight into programmed gel property control and has excellent potential for biomaterial and optoelectronic applications.
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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