Rapid and sensitive single-cell RNA sequencing with SHERRY2.

BACKGROUND: Prevalent single-cell transcriptomic profiling (scRNA-seq) methods are mainly based on the synthesis and enrichment of full-length double-stranded complementary DNA. These approaches are challenging to generate accurate quantification of transcripts when their abundance is low or their full-length amplifications are difficult. RESULTS: Based on our previous finding that Tn5 transposase can directly cut-and-tag DNA/RNA hetero-duplexes, we present SHERRY2, a specifically optimized protocol for scRNA-seq without second-strand cDNA synthesis. SHERRY2 is free of pre-amplification and eliminates the sequence-dependent bias. In comparison with other widely used scRNA-seq methods, SHERRY2 exhibits significantly higher sensitivity and accuracy even for single nuclei. Besides, SHERRY2 is simple and robust and can be easily scaled up to high-throughput experiments. When testing single lymphocytes and neuron nuclei, SHERRY2 not only obtained accurate countings of transcription factors and long non-coding RNAs, but also provided bias-free results that enriched genes in specific cellular components or functions, which outperformed other protocols. With a few thousand cells sequenced by SHERRY2, we confirmed the expression and dynamics of Myc in different cell types of germinal centers, which were previously only revealed by gene-specific amplification methods. CONCLUSIONS: SHERRY2 is able to provide high sensitivity, high accuracy, and high throughput for those applications that require a high number of genes identified in each cell. It can reveal the subtle transcriptomic difference between cells and facilitate important biological discoveries.

Natural diterpenoid EKO activates deubiqutinase ATXN3 to preserve vascular endothelial integrity and alleviate diabetic retinopathy through c-fos/focal adhesion axis.

Diabetic retinopathy (DR) is one of the most prevalent severe diabetic microvascular complications caused by hyperglycemia. Deciphering the underlying mechanism of vascular injury and finding ways to alleviate hyperglycemia induced microvascular complications is of great necessity. In this study, we identified that a compound ent-9α-hydroxy-15-oxo-16-kauren-19-oic acid (EKO), the diterpenoid isolated and purified from Pteris semipinnata L., exhibited good protective roles against vascular endothelial injury associated with diabetic retinopathy in vitro and in vivo. To further uncover the underlying mechanism, we used unbiased transcriptome sequencing analysis and showed substantial impairment in the focal adhesion pathway upon high glucose and IL-1ß stimulation. EKO could effectively improve endothelial focal adhesion pathway by enhancing the expression of two focal adhesion proteins Vinculin and ITGA11. We found that c-fos protein was involved in regulating the expression of Vinculin and ITGA11, a transcription factor component that was downregulated by high glucose and IL-1ß stimulation and recovered by EKO. Mechanically, EKO facilitated the binding of deubiquitylation enzyme ATXN3 to c-fos protein and promoted its deubiquitylation, thereby elevating its protein level to enhance the expression of Vinculin and ITGA11. Besides, EKO effectively suppressed ROS production and restored mitochondrial function. In vivo studies, we confirmed EKO could alleviate some of the indicators of diabetic mice. In addition, protein levels of ATXN3 and focal adhesion Vinculin molecule were also verified in vivo. Collectively, our findings addressed the endothelial protective role of natural diterpenoid EKO, with emphasize of mechanism on ATXN3/c-fos/focal adhesion signaling pathway as well as oxygen stress suppression, implicating its therapeutic potential in alleviating vascular endothelium injury and diabetic retinopathy.