Consensus prediction of cell type labels in single-cell data with popV.

Cell-type classification is a crucial step in single-cell sequencing analysis. Various methods have been proposed for transferring a cell-type label from an annotated reference atlas to unannotated query datasets. Existing methods for transferring cell-type labels lack proper uncertainty estimation for the resulting annotations, limiting interpretability and usefulness.

BICC1 Interacts with PKD1 and PKD2 to Drive Cystogenesis in ADPKD.

Background: Autosomal dominant polycystic kidney disease (ADPKD) is primarily of adult-onset and caused by pathogenic variants in or . Yet, disease expression is highly variable and includes very early-onset PKD presentations or infancy. In animal models, the RNA-binding molecule Bicc1 has been shown to play a crucial role in the pathogenesis of PKD.

Mapping protein-DNA interactions with DiMeLo-seq.

We recently developed directed methylation with long-read sequencing (DiMeLo-seq) to map protein-DNA interactions genome wide. DiMeLo-seq is capable of mapping multiple interaction sites on single DNA molecules, profiling protein binding in the context of endogenous DNA methylation, identifying haplotype-specific protein-DNA interactions and mapping protein-DNA interactions in repetitive regions of the genome that are difficult to study with short-read methods. With DiMeLo-seq, adenines in the vicinity of a protein of interest are methylated in situ by tethering the Hia5 methyltransferase to an antibody using protein A.

Deep generative modeling of transcriptional dynamics for RNA velocity analysis in single cells.

RNA velocity has been rapidly adopted to guide interpretation of transcriptional dynamics in snapshot single-cell data; however, current approaches for estimating RNA velocity lack effective strategies for quantifying uncertainty and determining the overall applicability to the system of interest. Here, we present veloVI (velocity variational inference), a deep generative modeling framework for estimating RNA velocity. veloVI learns a gene-specific dynamical model of RNA metabolism and provides a transcriptome-wide quantification of velocity uncertainty.

Single-cell multiomic analysis of thymocyte development reveals drivers of CD4 T cell and CD8 T cell lineage commitment.

The development of CD4 T cells and CD8 T cells in the thymus is critical to adaptive immunity and is widely studied as a model of lineage commitment. Recognition of self-peptide major histocompatibility complex (MHC) class I or II by the T cell antigen receptor (TCR) determines the CD8 or CD4 T cell lineage choice, respectively, but how distinct TCR signals drive transcriptional programs of lineage commitment remains largely unknown. Here we applied CITE-seq to measure RNA and surface proteins in thymocytes from wild-type and T cell lineage-restricted mice to generate a comprehensive timeline of cell states for each T cell lineage.

Loss of TJP1 disrupts gastrulation patterning and increases differentiation toward the germ cell lineage in human pluripotent stem cells.

Biological patterning events that occur early in development establish proper tissue morphogenesis. Identifying the mechanisms that guide these patterning events is necessary in order to understand the molecular drivers of development and disease and to build tissues in vitro. In this study, we use an in vitro model of gastrulation to study the role of tight junctions and apical/basolateral polarity in modulating bone morphogenic protein-4 (BMP4) signaling and gastrulation-associated patterning in colonies of human pluripotent stem cells (hPSCs).

Mapping the transcriptional landscape of human white and brown adipogenesis using single-nuclei RNA-seq.

Adipogenesis is key to maintaining organism-wide energy balance and healthy metabolic phenotype, making it critical to thoroughly comprehend its molecular regulation in humans. By single-nuclei RNA-sequencing (snRNA-seq) of over 20,000 differentiating white and brown preadipocytes, we constructed a high-resolution temporal transcriptional landscape of human white and brown adipogenesis. White and brown preadipocytes were isolated from a single individual's neck region, thereby eliminating inter-subject variability across two distinct lineages.

Towards an active droplet-based microfluidic platform for programmable fluid handling.

Droplet-based microfluidic systems have emerged as powerful alternatives to conventional high throughput screening platforms, due to their operational flexibility, high-throughput nature and ability to efficiently process small fluid volumes. However, the challenges associated with performing bespoke operations on user-defined droplets often limit their utility in screening applications that involve complex workflows. To this end, the marriage of droplet- and valve-based microfluidic technologies offers the prospect of balancing the controllability of droplet manipulations and analytical throughput.

The association of urinary epidermal growth factors with ADPKD disease severity and progression.

Background: The epidermal growth factor receptor (EGFR) pathway is involved in kidney tissue repair and growth. Preclinical interventional data and scarce human data have suggested a role for this pathway in the pathophysiology of autosomal dominant polycystic kidney disease (ADPKD), while other data have suggested that its activation is causally linked to repair of damaged kidney tissue. We hypothesize that urinary EGFR ligands, as a reflection of EGFR activity, are associated with kidney function decline in ADPKD in the context of tissue repair following injury, and as the disease progresses as a sign of insufficient repair.

Phototoxic effects of nonlinear optical microscopy on cell cycle, oxidative states, and gene expression.

Nonlinear optical imaging modalities, such as stimulated Raman scattering (SRS) microscopy, use pulsed-laser excitation with high peak intensity that can perturb the native state of cells. In this study, we used bulk RNA sequencing, quantitative measurement of cell proliferation, and fluorescent measurement of the generation of reactive oxygen species to assess phototoxic effects of near-IR pulsed laser radiation, at different time scales, for laser excitation settings relevant to SRS imaging. We define a range of laser excitation settings for which there was no significant change in mouse Neuro2A cells after laser exposure.

Histologically resolved multiomics enables precise molecular profiling of human intratumor heterogeneity.

Both the composition of cell types and their spatial distribution in a tissue play a critical role in cellular function, organ development, and disease progression. For example, intratumor heterogeneity and the distribution of transcriptional and genetic events in single cells drive the genesis and development of cancer. However, it can be challenging to fully characterize the molecular profile of cells in a tissue with high spatial resolution because microscopy has limited ability to extract comprehensive genomic information, and the spatial resolution of genomic techniques tends to be limited by dissection.

DiMeLo-seq: a long-read, single-molecule method for mapping protein-DNA interactions genome wide.

Studies of genome regulation routinely use high-throughput DNA sequencing approaches to determine where specific proteins interact with DNA, and they rely on DNA amplification and short-read sequencing, limiting their quantitative application in complex genomic regions. To address these limitations, we developed directed methylation with long-read sequencing (DiMeLo-seq), which uses antibody-tethered enzymes to methylate DNA near a target protein's binding sites in situ. These exogenous methylation marks are then detected simultaneously with endogenous CpG methylation on unamplified DNA using long-read, single-molecule sequencing technologies.

The complete sequence of a human genome.

Since its initial release in 2000, the human reference genome has covered only the euchromatic fraction of the genome, leaving important heterochromatic regions unfinished. Addressing the remaining 8% of the genome, the Telomere-to-Telomere (T2T) Consortium presents a complete 3.055 billion-base pair sequence of a human genome, T2T-CHM13, that includes gapless assemblies for all chromosomes except Y, corrects errors in the prior references, and introduces nearly 200 million base pairs of sequence containing 1956 gene predictions, 99 of which are predicted to be protein coding.

Complete genomic and epigenetic maps of human centromeres.

Existing human genome assemblies have almost entirely excluded repetitive sequences within and near centromeres, limiting our understanding of their organization, evolution, and functions, which include facilitating proper chromosome segregation. Now, a complete, telomere-to-telomere human genome assembly (T2T-CHM13) has enabled us to comprehensively characterize pericentromeric and centromeric repeats, which constitute 6.2% of the genome (189.

Mechanical phenotyping reveals unique biomechanical responses in retinoic acid-resistant acute promyelocytic leukemia.

All-trans retinoic acid (ATRA) is an essential therapy in the treatment of acute promyelocytic leukemia (APL), but nearly 20% of patients with APL are resistant to ATRA. As there are no biomarkers for ATRA resistance that yet exist, we investigated whether cell mechanics could be associated with this pathological phenotype. Using mechano-node-pore sensing, a single-cell mechanical phenotyping platform, and patient-derived APL cell lines, we discovered that ATRA-resistant APL cells are less mechanically pliable.

CXCR3 regulates stem and proliferative CD8+ T cells during chronic infection by promoting interactions with DCs in splenic bridging channels.

Production of effector CD8+ T cells during persistent infection requires a stable pool of stem-like cells that can give rise to effector cells via a proliferative intermediate population. In infection models marked by T cell exhaustion, this process can be transiently induced by checkpoint blockade but occurs spontaneously in mice chronically infected with the protozoan intracellular parasite Toxoplasma gondii. We observe distinct locations for parasite-specific T cell subsets, implying a link between differentiation and anatomical niches in the spleen.

Characterization of transcript enrichment and detection bias in single-nucleus RNA-seq for mapping of distinct human adipocyte lineages.

Single-cell RNA sequencing (scRNA-seq) enables molecular characterization of complex biological tissues at high resolution. The requirement of single-cell extraction, however, makes it challenging for profiling tissues such as adipose tissue, for which collection of intact single adipocytes is complicated by their fragile nature. For such tissues, single-nucleus extraction is often much more efficient and therefore single-nucleus RNA sequencing (snRNA-seq) presents an alternative to scRNA-seq.

Single cell biology-a Keystone Symposia report.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming.

T cell self-reactivity during thymic development dictates the timing of positive selection.

Functional tuning of T cells based on their degree of self-reactivity is established during positive selection in the thymus, although how positive selection differs for thymocytes with relatively low versus high self-reactivity is unclear. In addition, preselection thymocytes are highly sensitive to low-affinity ligands, but the mechanism underlying their enhanced T cell receptor (TCR) sensitivity is not fully understood. Here we show that murine thymocytes with low self-reactivity experience briefer TCR signals and complete positive selection more slowly than those with high self-reactivity.

Vascular smooth muscle-derived Trpv1 progenitors are a source of cold-induced thermogenic adipocytes.

Brown adipose tissue (BAT) and beige fat function in energy expenditure in part due to their role in thermoregulation, making these tissues attractive targets for treating obesity and metabolic disorders. While prolonged cold exposure promotes de novo recruitment of brown adipocytes, the exact sources of cold-induced thermogenic adipocytes are not completely understood. Here, we identify transient receptor potential cation channel subfamily V member 1 (Trpv1) vascular smooth muscle (VSM) cells as previously unidentified thermogenic adipocyte progenitors.

Paper-thin multilayer microfluidic devices with integrated valves.

Integrated valve microfluidics has an unparalleled capability to automate rapid delivery of fluids at the nanoliter scale for high-throughput biological experimentation. However, multilayer soft lithography, which is used to fabricate valve-microfluidics, produces devices with a minimum thickness of around five millimeters. This form-factor limitation prevents the use of such devices in experiments with limited sample thickness tolerance such as 4-pi microscopy, stimulated Raman scattering microscopy, and many forms of optical or magnetic tweezer applications.

Joint probabilistic modeling of single-cell multi-omic data with totalVI.

The paired measurement of RNA and surface proteins in single cells with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) is a promising approach to connect transcriptional variation with cell phenotypes and functions. However, combining these paired views into a unified representation of cell state is made challenging by the unique technical characteristics of each measurement. Here we present Total Variational Inference (totalVI; https://scvi-tools.