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Nature Methods

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https://www.readbyqxmd.com/read/28218900/in-vivo-three-photon-imaging-of-activity-of-gcamp6-labeled-neurons-deep-in-intact-mouse-brain
#1
Dimitre G Ouzounov, Tianyu Wang, Mengran Wang, Danielle D Feng, Nicholas G Horton, Jean C Cruz-Hernández, Yu-Ting Cheng, Jacob Reimer, Andreas S Tolias, Nozomi Nishimura, Chris Xu
High-resolution optical imaging is critical to understanding brain function. We demonstrate that three-photon microscopy at 1,300-nm excitation enables functional imaging of GCaMP6s-labeled neurons beyond the depth limit of two-photon microscopy. We record spontaneous activity from up to 150 neurons in the hippocampal stratum pyramidale at ∼1-mm depth within an intact mouse brain. Our method creates opportunities for noninvasive recording of neuronal activity with high spatial and temporal resolution deep within scattering brain tissues...
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28218899/prospective-identification-of-hematopoietic-lineage-choice-by-deep-learning
#2
Felix Buggenthin, Florian Buettner, Philipp S Hoppe, Max Endele, Manuel Kroiss, Michael Strasser, Michael Schwarzfischer, Dirk Loeffler, Konstantinos D Kokkaliaris, Oliver Hilsenbeck, Timm Schroeder, Fabian J Theis, Carsten Marr
Differentiation alters molecular properties of stem and progenitor cells, leading to changes in their shape and movement characteristics. We present a deep neural network that prospectively predicts lineage choice in differentiating primary hematopoietic progenitors using image patches from brightfield microscopy and cellular movement. Surprisingly, lineage choice can be detected up to three generations before conventional molecular markers are observable. Our approach allows identification of cells with differentially expressed lineage-specifying genes without molecular labeling...
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28218898/detecting-dna-cytosine-methylation-using-nanopore-sequencing
#3
Jared T Simpson, Rachael E Workman, P C Zuzarte, Matei David, L J Dursi, Winston Timp
In nanopore sequencing devices, electrolytic current signals are sensitive to base modifications, such as 5-methylcytosine (5-mC). Here we quantified the strength of this effect for the Oxford Nanopore Technologies MinION sequencer. By using synthetically methylated DNA, we were able to train a hidden Markov model to distinguish 5-mC from unmethylated cytosine. We applied our method to sequence the methylome of human DNA, without requiring special steps for library preparation.
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28218897/mapping-dna-methylation-with-high-throughput-nanopore-sequencing
#4
Arthur C Rand, Miten Jain, Jordan M Eizenga, Audrey Musselman-Brown, Hugh E Olsen, Mark Akeson, Benedict Paten
DNA chemical modifications regulate genomic function. We present a framework for mapping cytosine and adenosine methylation with the Oxford Nanopore Technologies MinION using this nanopore sequencer's ionic current signal. We map three cytosine variants and two adenine variants. The results show that our model is sensitive enough to detect changes in genomic DNA methylation levels as a function of growth phase in Escherichia coli.
February 20, 2017: Nature Methods
https://www.readbyqxmd.com/read/28192420/atomic-resolution-structures-from-fragmented-protein-crystals-with-the-cryoem-method-microed
#5
M Jason de la Cruz, Johan Hattne, Dan Shi, Paul Seidler, Jose Rodriguez, Francis E Reyes, Michael R Sawaya, Duilio Cascio, Simon C Weiss, Sun Kyung Kim, Cynthia S Hinck, Andrew P Hinck, Guillermo Calero, David Eisenberg, Tamir Gonen
Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography...
February 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/28192419/seq-well-portable-low-cost-rna-sequencing-of-single-cells-at-high-throughput
#6
Todd M Gierahn, Marc H Wadsworth, Travis K Hughes, Bryan D Bryson, Andrew Butler, Rahul Satija, Sarah Fortune, J Christopher Love, Alex K Shalek
Single-cell RNA-seq can precisely resolve cellular states, but applying this method to low-input samples is challenging. Here, we present Seq-Well, a portable, low-cost platform for massively parallel single-cell RNA-seq. Barcoded mRNA capture beads and single cells are sealed in an array of subnanoliter wells using a semipermeable membrane, enabling efficient cell lysis and transcript capture. We use Seq-Well to profile thousands of primary human macrophages exposed to Mycobacterium tuberculosis.
February 13, 2017: Nature Methods
https://www.readbyqxmd.com/read/28165473/cryosparc-algorithms-for-rapid-unsupervised-cryo-em-structure-determination
#7
Ali Punjani, John L Rubinstein, David J Fleet, Marcus A Brubaker
Single-particle electron cryomicroscopy (cryo-EM) is a powerful method for determining the structures of biological macromolecules. With automated microscopes, cryo-EM data can often be obtained in a few days. However, processing cryo-EM image data to reveal heterogeneity in the protein structure and to refine 3D maps to high resolution frequently becomes a severe bottleneck, requiring expert intervention, prior structural knowledge, and weeks of calculations on expensive computer clusters. Here we show that stochastic gradient descent (SGD) and branch-and-bound maximum likelihood optimization algorithms permit the major steps in cryo-EM structure determination to be performed in hours or minutes on an inexpensive desktop computer...
February 6, 2017: Nature Methods
https://www.readbyqxmd.com/read/28135259/building-proteometools-based-on-a-complete-synthetic-human-proteome
#8
Daniel P Zolg, Mathias Wilhelm, Karsten Schnatbaum, Johannes Zerweck, Tobias Knaute, Bernard Delanghe, Derek J Bailey, Siegfried Gessulat, Hans-Christian Ehrlich, Maximilian Weininger, Peng Yu, Judith Schlegl, Karl Kramer, Tobias Schmidt, Ulrike Kusebauch, Eric W Deutsch, Ruedi Aebersold, Robert L Moritz, Holger Wenschuh, Thomas Moehring, Stephan Aiche, Andreas Huhmer, Ulf Reimer, Bernhard Kuster
We describe ProteomeTools, a project building molecular and digital tools from the human proteome to facilitate biomedical research. Here we report the generation and multimodal liquid chromatography-tandem mass spectrometry analysis of >330,000 synthetic tryptic peptides representing essentially all canonical human gene products, and we exemplify the utility of these data in several applications. The resource (available at http://www.proteometools.org) will be extended to >1 million peptides, and all data will be shared with the community via ProteomicsDB and ProteomeXchange...
January 30, 2017: Nature Methods
https://www.readbyqxmd.com/read/28135258/sequencing-thousands-of-single-cell-genomes-with-combinatorial-indexing
#9
Sarah A Vitak, Kristof A Torkenczy, Jimi L Rosenkrantz, Andrew J Fields, Lena Christiansen, Melissa H Wong, Lucia Carbone, Frank J Steemers, Andrew Adey
Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants...
January 30, 2017: Nature Methods
https://www.readbyqxmd.com/read/28135257/one-step-generation-of-conditional-and-reversible-gene-knockouts
#10
Amanda Andersson-Rolf, Roxana C Mustata, Alessandra Merenda, Jihoon Kim, Sajith Perera, Tiago Grego, Katie Andrews, Katie Tremble, José C R Silva, Juergen Fink, William C Skarnes, Bon-Kyoung Koo
Loss-of-function studies are key for investigating gene function, and CRISPR technology has made genome editing widely accessible in model organisms and cells. However, conditional gene inactivation in diploid cells is still difficult to achieve. Here, we present CRISPR-FLIP, a strategy that provides an efficient, rapid and scalable method for biallelic conditional gene knockouts in diploid or aneuploid cells, such as pluripotent stem cells, 3D organoids and cell lines, by co-delivery of CRISPR-Cas9 and a universal conditional intronic cassette...
January 30, 2017: Nature Methods
https://www.readbyqxmd.com/read/28135256/characterizing-cell-subsets-using-marker-enrichment-modeling
#11
Kirsten E Diggins, Allison R Greenplate, Nalin Leelatian, Cara E Wogsland, Jonathan M Irish
Learning cell identity from high-content single-cell data presently relies on human experts. We present marker enrichment modeling (MEM), an algorithm that objectively describes cells by quantifying contextual feature enrichment and reporting a human- and machine-readable text label. MEM outperforms traditional metrics in describing immune and cancer cell subsets from fluorescence and mass cytometry. MEM provides a quantitative language to communicate characteristics of new and established cytotypes observed in complex tissues...
January 30, 2017: Nature Methods
https://www.readbyqxmd.com/read/28135255/massively-multiplex-single-cell-hi-c
#12
Vijay Ramani, Xinxian Deng, Ruolan Qiu, Kevin L Gunderson, Frank J Steemers, Christine M Disteche, William S Noble, Zhijun Duan, Jay Shendure
We present single-cell combinatorial indexed Hi-C (sciHi-C), a method that applies combinatorial cellular indexing to chromosome conformation capture. In this proof of concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karyotypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation. Our results demonstrate that combinatorial indexing is a generalizable strategy for single-cell genomics...
January 30, 2017: Nature Methods
https://www.readbyqxmd.com/read/28114289/optogenetic-inhibition-of-behavior-with-anion-channelrhodopsins
#13
Farhan Mohammad, James C Stewart, Stanislav Ott, Katarina Chlebikova, Jia Yi Chua, Tong-Wey Koh, Joses Ho, Adam Claridge-Chang
Optogenetics uses light exposure to manipulate physiology in genetically modified organisms. Abundant tools for optogenetic excitation are available, but the limitations of current optogenetic inhibitors present an obstacle to demonstrating the necessity of neuronal circuits. Here we show that anion channelrhodopsins can be used to specifically and rapidly inhibit neural systems involved in Drosophila locomotion, wing expansion, memory retrieval and gustation, thus demonstrating their broad utility in the circuit analysis of behavior...
January 23, 2017: Nature Methods
https://www.readbyqxmd.com/read/28114288/high-fidelity-mass-analysis-unveils-heterogeneity-in-intact-ribosomal-particles
#14
Michiel van de Waterbeemd, Kyle L Fort, Dmitriy Boll, Maria Reinhardt-Szyba, Andrew Routh, Alexander Makarov, Albert J R Heck
Investigation of the structure, assembly and function of protein-nucleic acid macromolecular machines requires multidimensional molecular and structural biology approaches. We describe modifications to an Orbitrap mass spectrometer, enabling high-resolution native MS analysis of 0.8- to 2.3-MDa prokaryotic 30S, 50S and 70S ribosome particles and the 9-MDa Flock House virus. The instrument's improved mass range and sensitivity readily exposes unexpected binding of the ribosome-associated protein SRA.
January 23, 2017: Nature Methods
https://www.readbyqxmd.com/read/28114287/single-cell-mrna-quantification-and-differential-analysis-with-census
#15
Xiaojie Qiu, Andrew Hill, Jonathan Packer, Dejun Lin, Yi-An Ma, Cole Trapnell
Single-cell gene expression studies promise to reveal rare cell types and cryptic states, but the high variability of single-cell RNA-seq measurements frustrates efforts to assay transcriptional differences between cells. We introduce the Census algorithm to convert relative RNA-seq expression levels into relative transcript counts without the need for experimental spike-in controls. Analyzing changes in relative transcript counts led to dramatic improvements in accuracy compared to normalized read counts and enabled new statistical tests for identifying developmentally regulated genes...
January 23, 2017: Nature Methods
https://www.readbyqxmd.com/read/28099430/pooled-crispr-screening-with-single-cell-transcriptome-readout
#16
Paul Datlinger, André F Rendeiro, Christian Schmidl, Thomas Krausgruber, Peter Traxler, Johanna Klughammer, Linda C Schuster, Amelie Kuchler, Donat Alpar, Christoph Bock
CRISPR-based genetic screens are accelerating biological discovery, but current methods have inherent limitations. Widely used pooled screens are restricted to simple readouts including cell proliferation and sortable marker proteins. Arrayed screens allow for comprehensive molecular readouts such as transcriptome profiling, but at much lower throughput. Here we combine pooled CRISPR screening with single-cell RNA sequencing into a broadly applicable workflow, directly linking guide RNA expression to transcriptome responses in thousands of individual cells...
January 18, 2017: Nature Methods
https://www.readbyqxmd.com/read/28092692/smile-seq-identifies-binding-motifs-of-single-and-dimeric-transcription-factors
#17
Alina Isakova, Romain Groux, Michael Imbeault, Pernille Rainer, Daniel Alpern, Riccardo Dainese, Giovanna Ambrosini, Didier Trono, Philipp Bucher, Bart Deplancke
Resolving the DNA-binding specificities of transcription factors (TFs) is of critical value for understanding gene regulation. Here, we present a novel, semiautomated protein-DNA interaction characterization technology, selective microfluidics-based ligand enrichment followed by sequencing (SMiLE-seq). SMiLE-seq is neither limited by DNA bait length nor biased toward strong affinity binders; it probes the DNA-binding properties of TFs over a wide affinity range in a fast and cost-effective fashion. We validated SMiLE-seq by analyzing 58 full-length human, mouse, and Drosophila TFs from distinct structural classes...
January 16, 2017: Nature Methods
https://www.readbyqxmd.com/read/28092691/effective-detection-of-variation-in-single-cell-transcriptomes-using-matq-seq
#18
Kuanwei Sheng, Wenjian Cao, Yichi Niu, Qing Deng, Chenghang Zong
The quantification of transcriptional variation in single cells, particularly within the same cell population, is currently limited by the low sensitivity and high technical noise of single-cell RNA-seq assays. We report multiple annealing and dC-tailing-based quantitative single-cell RNA-seq (MATQ-seq), a highly sensitive and quantitative method for single-cell sequencing of total RNA. By systematically determining technical noise, we show that MATQ-seq captures genuine biological variation between whole transcriptomes of single cells...
January 16, 2017: Nature Methods
https://www.readbyqxmd.com/read/28092690/post-translational-selective-intracellular-silencing-of-acetylated-proteins-with-de-novo-selected-intrabodies
#19
Michele Chirichella, Simonetta Lisi, Marco Fantini, Martina Goracci, Mariantonietta Calvello, Rossella Brandi, Ivan Arisi, Mara D'Onofrio, Cristina Di Primio, Antonino Cattaneo
The ability to selectively interfere with post-translationally modified proteins would have many biological and therapeutic applications. However, post-translational modifications cannot be selectively targeted by nucleic-acid-based interference approaches. Here we describe post-translational intracellular silencing antibody technology (PISA), a method for selecting intrabodies against post-translationally modified proteins. We demonstrate our method by generating intrabodies against native acetylated proteins and showing functional interference in living cells...
January 16, 2017: Nature Methods
https://www.readbyqxmd.com/read/28068316/scalable-whole-genome-single-cell-library-preparation-without-preamplification
#20
Hans Zahn, Adi Steif, Emma Laks, Peter Eirew, Michael VanInsberghe, Sohrab P Shah, Samuel Aparicio, Carl L Hansen
Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations...
January 9, 2017: Nature Methods
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