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SummaryTranscription is a highly dynamic process. Consequently, we have developed native elongating transcript sequencing technology for mammalian chromatin (mNET-seq), which generates single-nucleotide resolution, nascent transcription profiles. Nascent RNA was detected in the active site of RNA polymerase II (Pol II) along with associated RNA processing intermediates. In particular, we detected 5′splice site cleavage by the spliceosome, showing that cleaved upstream exon transcripts are associated with Pol II CTD phosphorylated on the serine 5 position (S5P), which is accumulated over downstream exons. Also, depletion of termination factors substantially reduces Pol II pausing at gene ends, leading to termination defects. Notably, termination factors play an additional promoter role by restricting non-productive RNA synthesis in a Pol II CTD S2P-specific manner. Our results suggest that CTD phosphorylation patterns established for yeast transcription are significantly different in mammals. Taken together, mNET-seq provides dynamic and detailed snapshots of the complex events underlying transcription in mammals.

Droplet-based single-cell RNA sequencing protocols have dramatically increased the throughput of single-cell transcriptomics studies. A key computational challenge when processing these data is to distinguish libraries for real cells from empty droplets. Here, we describe a new statistical method for calling cells from droplet-based data, based on detecting significant deviations from the expression profile of the ambient solution. Using simulations, we demonstrate that EmptyDrops has greater power than existing approaches while controlling the false discovery rate among detected cells. Our method also retains distinct cell types that would have been discarded by existing methods in several real data sets.Electronic supplementary materialThe online version of this article (10.1186/s13059-019-1662-y) contains supplementary material, which is available to authorized users.

SummaryNumerous long intervening noncoding RNAs (lincRNAs) are generated from the mammalian genome by RNA polymerase II (Pol II) transcription. Although multiple functions have been ascribed to lincRNAs, their synthesis and turnover remain poorly characterized. Here, we define systematic differences in transcription and RNA processing between protein-coding and lincRNA genes in human HeLa cells. This is based on a range of nascent transcriptomic approaches applied to different nuclear fractions, including mammalian native elongating transcript sequencing (mNET-seq). Notably, mNET-seq patterns specific for different Pol II CTD phosphorylation states reveal weak co-transcriptional splicing and poly(A) signal-independent Pol II termination of lincRNAs as compared to pre-mRNAs. In addition, lincRNAs are mostly restricted to chromatin, since they are rapidly degraded by the RNA exosome. We also show that a lincRNA-specific co-transcriptional RNA cleavage mechanism acts to induce premature termination. In effect, functional lincRNAs must escape from this targeted nuclear surveillance process.

Summary Non-lymphoid tissues (NLTs) harbor a pool of adaptive immune cells with largely unexplored phenotype and development. We used single-cell RNA-seq to characterize 35,000 CD4 + regulatory (Treg) and memory (Tmem) T cells in mouse skin and colon, their respective draining lymph nodes (LNs) and spleen. In these tissues, we identified Treg cell subpopulations with distinct degrees of NLT phenotype. Subpopulation pseudotime ordering and gene kinetics were consistent in recruitment to skin and colon, yet the initial NLT-priming in LNs and the final stages of NLT functional adaptation reflected tissue-specific differences. Predicted kinetics were recapitulated using an in vivo melanoma-induction model, validating key regulators and receptors. Finally, we profiled human blood and NLT Treg and Tmem cells, and identified cross-mammalian conserved tissue signatures. In summary, we describe the relationship between Treg cell heterogeneity and recruitment to NLTs through the combined use of computational prediction and in vivo validation.

Despite their crucial role in health and disease, our knowledge of immune cells within human tissues remains limited. We surveyed the immune compartment of 16 tissues from 12 adult donors by single-cell RNA sequencing and VDJ sequencing generating a dataset of ~360,000 cells. To systematically resolve immune cell heterogeneity across tissues, we developed CellTypist, a machine learning tool for rapid and precise cell type annotation. Using this approach, combined with detailed curation, we determined the tissue distribution of finely phenotyped immune cell types, revealing hitherto unappreciated tissue-specific features and clonal architecture of T and B cells. Our multitissue approach lays the foundation for identifying highly resolved immune cell types by leveraging a common reference dataset, tissue-integrated expression analysis, and antigen receptor sequencing.

Summary As the first line of defence against pathogens, cells mount an innate immune response, which is highly variable from cell to cell. The response must be potent yet carefully controlled to avoid self-damage. How these constraints have shaped the evolution of innate immunity remains poorly understood. Here, we characterise this programme’s transcriptional divergence between species and expression variability across cells. Using bulk and single-cell transcriptomics in fibroblasts and mononuclear phagocytes from different species, challenged with immune stimuli, we reveal a striking architecture of the innate immune response. Transcriptionally diverging genes, including cytokines and chemokines, vary across cells and have distinct promoter structures. Conversely, genes involved in response regulation, such as transcription factors and kinases, are conserved between species and display low cell-to-cell expression variability. We suggest that this unique expression pattern, observed across species and conditions, has evolved as a mechanism for fine-tuned regulation, achieving an effective but balanced response.

Gastrointestinal microbiota and immune cells interact closely and display regional specificity, but little is known about how these communities differ with location. Here, we simultaneously assess microbiota and single immune cells across the healthy, adult human colon, with paired characterization of immune cells in the mesenteric lymph nodes, to delineate colonic immune niches at steady-state. We describe distinct T helper cell activation and migration profiles along the colon and characterize the transcriptional adaptation trajectory of T regulatory cells between lymphoid tissue and colon. Finally, we show increasing B cell accumulation, clonal expansion and mutational frequency from cecum to sigmoid colon, and link this to the increasing number of reactive bacterial species.