Publications by authors named "J Shendure"

343 Publications

The landscape of alternative polyadenylation in single cells of the developing mouse embryo.

Nat Commun 2021 08 24;12(1):5101. Epub 2021 Aug 24.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

3' untranslated regions (3' UTRs) post-transcriptionally regulate mRNA stability, localization, and translation rate. While 3'-UTR isoforms have been globally quantified in limited cell types using bulk measurements, their differential usage among cell types during mammalian development remains poorly characterized. In this study, we examine a dataset comprising ~2 million nuclei spanning E9.5-E13.5 of mouse embryonic development to quantify transcriptome-wide changes in alternative polyadenylation (APA). We observe a global lengthening of 3' UTRs across embryonic stages in all cell types, although we detect shorter 3' UTRs in hematopoietic lineages and longer 3' UTRs in neuronal cell types within each stage. An analysis of RNA-binding protein (RBP) dynamics identifies ELAV-like family members, which are concomitantly induced in neuronal lineages and developmental stages experiencing 3'-UTR lengthening, as putative regulators of APA. By measuring 3'-UTR isoforms in an expansive single cell dataset, our work provides a transcriptome-wide and organism-wide map of the dynamic landscape of alternative polyadenylation during mammalian organogenesis.
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http://dx.doi.org/10.1038/s41467-021-25388-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8385098PMC
August 2021

SwabExpress: An end-to-end protocol for extraction-free covid-19 testing.

Clin Chem 2021 Jul 21. Epub 2021 Jul 21.

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

Background: The urgent need for massively scaled clinical testing for SARS-CoV-2, along with global shortages of critical reagents and supplies, has necessitated development of streamlined laboratory testing protocols. Conventional nucleic acid testing for SARS-CoV-2 involves collection of a clinical specimen with a nasopharyngeal swab in transport medium, nucleic acid extraction, and quantitative reverse transcription PCR (RT-qPCR) (1). As testing has scaled across the world, the global supply chain has buckled, rendering testing reagents and materials scarce (2). To address shortages, we developed SwabExpress, an end-to-end protocol developed to employ mass produced anterior nares swabs and bypass the requirement for transport media and nucleic acid extraction.

Methods: We evaluated anterior nares swabs, transported dry and eluted in low-TE buffer as a direct-to-RT-qPCR alternative to extraction-dependent viral transport media. We validated our protocol of using heat treatment for viral inactivation and added a proteinase K digestion step to reduce amplification interference. We tested this protocol across archived and prospectively collected swab specimens to fine-tune test performance.

Results: After optimization, SwabExpress has a low limit of detection at 2-4 molecules/uL, 100% sensitivity, and 99.4% specificity when compared side-by-side with a traditional RT-qPCR protocol employing extraction. On real-world specimens, SwabExpress outperforms an automated extraction system while simultaneously reducing cost and hands-on time.

Conclusion: SwabExpress is a simplified workflow that facilitates scaled testing for COVID-19 without sacrificing test performance. It may serve as a template for the simplification of PCR-based clinical laboratory tests, particularly in times of critical shortages during pandemics.
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http://dx.doi.org/10.1093/clinchem/hvab132DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8406859PMC
July 2021

Embryo-scale, single-cell spatial transcriptomics.

Science 2021 07;373(6550):111-117

Department of Genome Sciences, University of Washington, Seattle, WA, USA.

Spatial patterns of gene expression manifest at scales ranging from local (e.g., cell-cell interactions) to global (e.g., body axis patterning). However, current spatial transcriptomics methods either average local contexts or are restricted to limited fields of view. Here, we introduce sci-Space, which retains single-cell resolution while resolving spatial heterogeneity at larger scales. Applying sci-Space to developing mouse embryos, we captured approximate spatial coordinates and whole transcriptomes of about 120,000 nuclei. We identify thousands of genes exhibiting anatomically patterned expression, leverage spatial information to annotate cellular subtypes, show that cell types vary substantially in their extent of spatial patterning, and reveal correlations between pseudotime and the migratory patterns of differentiating neurons. Looking forward, we anticipate that sci-Space will facilitate the construction of spatially resolved single-cell atlases of mammalian development.
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http://dx.doi.org/10.1126/science.abb9536DOI Listing
July 2021

Benchmarked approaches for reconstruction of in vitro cell lineages and in silico models of C. elegans and M. musculus developmental trees.

Cell Syst 2021 Aug 18;12(8):810-826.e4. Epub 2021 Jun 18.

Program in Quantitative and Computational Biosciences, Baylor College of Medicine, Houston, TX 77030, USA.

The recent advent of CRISPR and other molecular tools enabled the reconstruction of cell lineages based on induced DNA mutations and promises to solve the ones of more complex organisms. To date, no lineage reconstruction algorithms have been rigorously examined for their performance and robustness across dataset types and number of cells. To benchmark such methods, we decided to organize a DREAM challenge using in vitro experimental intMEMOIR recordings and in silico data for a C. elegans lineage tree of about 1,000 cells and a Mus musculus tree of 10,000 cells. Some of the 22 approaches submitted had excellent performance, but structural features of the trees prevented optimal reconstructions. Using smaller sub-trees as training sets proved to be a good approach for tuning algorithms to reconstruct larger trees. The simulation and reconstruction methods here generated delineate a potential way forward for solving larger cell lineage trees such as in mouse.
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http://dx.doi.org/10.1016/j.cels.2021.05.008DOI Listing
August 2021

Single-cell lineage tracing of metastatic cancer reveals selection of hybrid EMT states.

Cancer Cell 2021 Aug 10;39(8):1150-1162.e9. Epub 2021 Jun 10.

Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA; Department of Cell & Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA; Institute for Regenerative Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

The underpinnings of cancer metastasis remain poorly understood, in part due to a lack of tools for probing their emergence at high resolution. Here we present macsGESTALT, an inducible CRISPR-Cas9-based lineage recorder with highly efficient single-cell capture of both transcriptional and phylogenetic information. Applying macsGESTALT to a mouse model of metastatic pancreatic cancer, we recover ∼380,000 CRISPR target sites and reconstruct dissemination of ∼28,000 single cells across multiple metastatic sites. We find that cells occupy a continuum of epithelial-to-mesenchymal transition (EMT) states. Metastatic potential peaks in rare, late-hybrid EMT states, which are aggressively selected from a predominately epithelial ancestral pool. The gene signatures of these late-hybrid EMT states are predictive of reduced survival in both human pancreatic and lung cancer patients, highlighting their relevance to clinical disease progression. Finally, we observe evidence for in vivo propagation of S100 family gene expression across clonally distinct metastatic subpopulations.
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http://dx.doi.org/10.1016/j.ccell.2021.05.005DOI Listing
August 2021
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