SMURF Reconstructs Single-Cells from Visium HD Data to Reveal Zonation of Transcriptional Programs in the Intestine.

High-resolution spatial transcriptomics demands new computational tools to accurately assign transcripts to individual cells. We developed SMURF (Segmentation and Manifold UnRolling Framework), a novel soft-segmentation algorithm that maps mRNAs from barcoded capture spots to nearby nuclei. SMURF also "unrolls" cells in complex tissue structures, placing them on standard Cartesian coordinates, thus empowering studies of cellular organization in tissues.

EGR1 drives sex-differences in glioblastoma tumorigenicity.

Although progress has been made in treating glioblastoma (GBM), with fewer than 5% of patients surviving more than 5 years after diagnosis. For reasons that are not well understood, females are roughly 60% as likely as males to develop GBM, and female patients consistently respond better to treatment than do males. Understanding the molecular etiology of these sex differences in tumor progression and resiliency to treatment could reveal potent new therapeutic targets, ultimately improving survival of both male and female GBM patients.

Whole-Genome Sequencing Reveals Individual and Cohort Level Insights into Chromosome 9p Syndromes.

Previous genomic efforts on chromosome 9p deletion and duplication syndromes have utilized low resolution strategies (i.e., karyotypes, chromosome microarrays).

Prior epigenetic status predicts future susceptibility to seizures in mice.

Wide variation of responses to identical stimuli presented to genetically inbred mice suggests the hypothesis that stochastic epigenetic variation during neurodevelopment can mediate such phenotypic differences. However, this hypothesis is largely untested since capturing pre-existing molecular states requires non-destructive, longitudinal recording. Therefore, we tested the potential of Calling Cards (CC) to record transient neuronal enhancer activity during postnatal development, and thereby associate epigenetic variation with a subsequent phenotypic presentation - degree of seizure response to the pro-convulsant pentylenetetrazol.

MYT1L deficiency impairs excitatory neuron trajectory during cortical development.

Mutations reducing the function of MYT1L, a neuron-specific transcription factor, are associated with a syndromic neurodevelopmental disorder. MYT1L is used as a pro-neural factor in fibroblast-to-neuron transdifferentiation and is hypothesized to influence neuronal specification and maturation, but it is not clear which neuron types are most impacted by MYT1L loss. In this study, we profile 412,132 nuclei from the forebrains of wild-type and MYT1L-deficient mice at three developmental stages: E14 at the peak of neurogenesis, P1 when cortical neurons have been born, and P21 when neurons are maturing, to examine the role of MYT1L levels on neuronal development.

MYT1L deficiency impairs excitatory neuron trajectory during cortical development.

Mutations that reduce the function of MYT1L, a neuron-specific transcription factor, are associated with a syndromic neurodevelopmental disorder. Furthermore, MYT1L is routinely used as a proneural factor in fibroblast-to-neuron transdifferentiation. MYT1L has been hypothesized to play a role in the trajectory of neuronal specification and subtype specific maturation, but this hypothesis has not been directly tested, nor is it clear which neuron types are most impacted by MYT1L loss.

Pycallingcards: an integrated environment for visualizing, analyzing, and interpreting Calling Cards data.

Motivation: Unraveling the transcriptional programs that control how cells divide, differentiate, and respond to their environments requires a precise understanding of transcription factors' (TFs) DNA-binding activities. Calling cards (CC) technology uses transposons to capture transient TF binding events at one instant in time and then read them out at a later time. This methodology can also be used to simultaneously measure TF binding and mRNA expression from single-cell CC and to record and integrate TF binding events across time in any cell type of interest without the need for purification.

Calling Cards: A Customizable Platform to Longitudinally Record Protein-DNA Interactions Over Time in Cells and Tissues.

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next-generation sequencing. Compared with other genomic assays, readouts of which provide a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes.

Epigenetic regulation of hybrid epithelial-mesenchymal cell states in cancer.

Epithelial-to-mesenchymal transition (EMT) is a process by which cells lose their epithelial characteristics and gain mesenchymal phenotypes. In cancer, EMT is thought to drive tumor invasion and metastasis. Recent efforts to understand EMT biology have uncovered that cells undergoing EMT attain a spectrum of intermediate "hybrid E/M" states, which exist along an epithelial-mesenchymal continuum.

Calling Cards: a customizable platform to longitudinally record protein-DNA interactions over time in cells and tissues.

Calling Cards is a platform technology to record a cumulative history of transient protein-DNA interactions in the genome of genetically targeted cell types. The record of these interactions is recovered by next generation sequencing. Compared to other genomic assays, whose readout provides a snapshot at the time of harvest, Calling Cards enables correlation of historical molecular states to eventual outcomes or phenotypes.

Zinc cluster transcription factors frequently activate target genes using a non-canonical half-site binding mode.

Gene expression changes are orchestrated by transcription factors (TFs), which bind to DNA to regulate gene expression. It remains surprisingly difficult to predict basic features of the transcriptional process, including in vivo TF occupancy. Existing thermodynamic models of TF function are often not concordant with experimental measurements, suggesting undiscovered biology.

A Suite of New Strain Construction Vectors for Gene Expression Knockdown in Budding Yeast.

Numerous tools for gene expression knockdown have been developed and characterized in the model organism and extended to facilitate studies in multicellular models. To comparatively evaluate the efficacy of these approaches, we systematically applied seven such published constitutive and inducible knockdown strategies to a panel of essential genes encoding nuclear-localized proteins. In this effort, we created the CEAS (C-SWAT for Essential Allele Strains) collection, a suite of tagging vectors for improved utility and ease of strain construction.

Pooled image-base screening of mitochondria with microraft isolation distinguishes pathogenic mitofusin 2 mutations.

Most human genetic variation is classified as variants of uncertain significance. While advances in genome editing have allowed innovation in pooled screening platforms, many screens deal with relatively simple readouts (viability, fluorescence) and cannot identify the complex cellular phenotypes that underlie most human diseases. In this paper, we present a generalizable functional genomics platform that combines high-content imaging, machine learning, and microraft isolation in a method termed "Raft-Seq".

Measuring transcription factor binding and gene expression using barcoded self-reporting transposon calling cards and transcriptomes.

Calling cards technology using self-reporting transposons enables the identification of DNA-protein interactions through RNA sequencing. Although immensely powerful, current implementations of calling cards in bulk experiments on populations of cells are technically cumbersome and require many replicates to identify independent insertions into the same genomic locus. Here, we have drastically reduced the cost and labor requirements of calling card experiments in bulk populations of cells by introducing a DNA barcode into the calling card itself.

Disentangling glial diversity in peripheral nerves at single-nuclei resolution.

The peripheral nerve contains diverse cell types that support its proper function and maintenance. In this study, we analyzed multiple peripheral nerves using single-nuclei RNA sequencing, which allowed us to circumvent difficulties encountered in analyzing cells with complex morphologies via conventional single-cell methods. The resultant mouse peripheral nerve cell atlas highlights a diversity of cell types, including multiple subtypes of Schwann cells (SCs), immune cells and stromal cells.

From karyotypes to precision genomics in 9p deletion and duplication syndromes.

While 9p deletion and duplication syndromes have been studied for several years, small sample sizes and minimal high-resolution data have limited a comprehensive delineation of genotypic and phenotypic characteristics. In this study, we examined genetic data from 719 individuals in the worldwide 9p Network Cohort: a cohort seven to nine times larger than any previous study of 9p. Most breakpoints occur in bands 9p22 and 9p24, accounting for 35% and 38% of all breakpoints, respectively.

Brd4-bound enhancers drive cell-intrinsic sex differences in glioblastoma.

Sex can be an important determinant of cancer phenotype, and exploring sex-biased tumor biology holds promise for identifying novel therapeutic targets and new approaches to cancer treatment. In an established isogenic murine model of glioblastoma (GBM), we discovered correlated transcriptome-wide sex differences in gene expression, H3K27ac marks, large Brd4-bound enhancer usage, and Brd4 localization to Myc and p53 genomic binding sites. These sex-biased gene expression patterns were also evident in human glioblastoma stem cells (GSCs).

High-resolution HLA typing by long reads from the R10.3 Oxford nanopore flow cells.

Nanopore sequencing has been investigated as a rapid and cost-efficient option for HLA typing in recent years. Despite the lower raw read accuracy, encouraging typing accuracy has been reported, and long reads from the platform offer additional benefits of the improved phasing of distant variants. The newly released R10.

Rapid and Extraction-Free Detection of SARS-CoV-2 from Saliva by Colorimetric Reverse-Transcription Loop-Mediated Isothermal Amplification.

Background: Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold-standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment, instrumentation, and labor.

Methods: To overcome these challenges, we developed a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step.

The qBED track: a novel genome browser visualization for point processes.

Summary: Transposon calling cards is a genomic assay for identifying transcription factor binding sites in both bulk and single cell experiments. Here, we describe the qBED format, an open, text-based standard for encoding and analyzing calling card data. In parallel, we introduce the qBED track on the WashU Epigenome Browser, a novel visualization that enables researchers to inspect calling card data in their genomic context.

High-throughput single-cell functional elucidation of neurodevelopmental disease-associated genes reveals convergent mechanisms altering neuronal differentiation.

The overwhelming success of exome- and genome-wide association studies in discovering thousands of disease-associated genes necessitates developing novel high-throughput functional genomics approaches to elucidate the molecular mechanisms of these genes. Here, we have coupled multiplexed repression of neurodevelopmental disease-associated genes to single-cell transcriptional profiling in differentiating human neurons to rapidly assay the functions of multiple genes in a disease-relevant context, assess potentially convergent mechanisms, and prioritize genes for specific functional assays. For a set of 13 autism spectrum disorder (ASD)-associated genes, we show that this approach generated important mechanistic insights, revealing two functionally convergent modules of ASD genes: one that delays neuron differentiation and one that accelerates it.

Human L1 Transposition Dynamics Unraveled with Functional Data Analysis.

Long INterspersed Elements-1 (L1s) constitute >17% of the human genome and still actively transpose in it. Characterizing L1 transposition across the genome is critical for understanding genome evolution and somatic mutations. However, to date, L1 insertion and fixation patterns have not been studied comprehensively.

Self-Reporting Transposons Enable Simultaneous Readout of Gene Expression and Transcription Factor Binding in Single Cells.

Cellular heterogeneity confounds in situ assays of transcription factor (TF) binding. Single-cell RNA sequencing (scRNA-seq) deconvolves cell types from gene expression, but no technology links cell identity to TF binding sites (TFBS) in those cell types. We present self-reporting transposons (SRTs) and use them in single-cell calling cards (scCC), a novel assay for simultaneously measuring gene expression and mapping TFBS in single cells.

Rapid and extraction-free detection of SARS-CoV-2 from saliva with colorimetric LAMP.

Rapid, reliable, and widespread testing is required to curtail the ongoing COVID-19 pandemic. Current gold standard nucleic acid tests are hampered by supply shortages in critical reagents including nasal swabs, RNA extraction kits, personal protective equipment (PPE), instrumentation, and labor. Here we present an approach to overcome these challenges with the development of a rapid colorimetric assay using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) optimized on human saliva samples without an RNA purification step.