Micropilot: automation of fluorescence microscopy–based imaging for systems biology

Micropilot: automation of fluorescence microscopy–based imaging for systems biology: "

Micropilot: automation of fluorescence microscopy–based imaging for systems biology

Nature Methods 8, 246 (2011). doi:10.1038/nmeth.1558

Authors: Christian Conrad, Annelie Wünsche, Tze Heng Tan, Jutta Bulkescher, Frank Sieckmann, Fatima Verissimo, Arthur Edelstein, Thomas Walter, Urban Liebel, Rainer Pepperkok & Jan Ellenberg

Quantitative microscopy relies on imaging of large cell numbers but is often hampered by time-consuming manual selection of specific cells. The 'Micropilot' software automatically detects cells of interest and launches complex imaging experiments including three-dimensional multicolor time-lapse or fluorescence recovery after photobleaching in live cells. In three independent experimental setups this allowed us to statistically analyze biological processes in detail and is thus a powerful tool for systems biology.

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The draft genome of the parasitic nematode Trichinella spiralis

The draft genome of the parasitic nematode Trichinella spiralis: "

The draft genome of the parasitic nematode Trichinella spiralis

Nature Genetics 43, 228 (2011). doi:10.1038/ng.769

Authors: Makedonka Mitreva, Douglas P Jasmer, Dante S Zarlenga, Zhengyuan Wang, Sahar Abubucker, John Martin, Christina M Taylor, Yong Yin, Lucinda Fulton, Pat Minx, Shiaw-Pyng Yang, Wesley C Warren, Robert S Fulton, Veena Bhonagiri, Xu Zhang, Kym Hallsworth-Pepin, Sandra W Clifton, James P McCarter, Judith Appleton, Elaine R Mardis & Richard K Wilson

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Chromatin accessibility pre-determines glucocorticoid receptor binding patterns

Chromatin accessibility pre-determines glucocorticoid receptor binding patterns: "

Chromatin accessibility pre-determines glucocorticoid receptor binding patterns

Nature Genetics 43, 264 (2011). doi:10.1038/ng.759

Authors: Sam John, Peter J Sabo, Robert E Thurman, Myong-Hee Sung, Simon C Biddie, Thomas A Johnson, Gordon L Hager & John A Stamatoyannopoulos

Development, differentiation and response to environmental stimuli are characterized by sequential changes in cellular state initiated by the de novo binding of regulated transcriptional factors to their cognate genomic sites. The mechanism whereby a given regulatory factor selects a limited number of in vivo targets from a myriad of potential genomic binding sites is undetermined. Here we show that up to 95% of de novo genomic binding by the glucocorticoid receptor, a paradigmatic ligand-activated transcription factor, is targeted to preexisting foci of accessible chromatin. Factor binding invariably potentiates chromatin accessibility. Cell-selective glucocorticoid receptor occupancy patterns appear to be comprehensively predetermined by cell-specific differences in baseline chromatin accessibility patterns, with secondary contributions from local sequence features. The results define a framework for understanding regulatory factor–genome interactions and provide a molecular basis for the tissue selectivity of steroid pharmaceuticals and other agents that intersect the living genome.

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Discovery and genotyping of genome structural polymorphism by sequencing on a population scale

Discovery and genotyping of genome structural polymorphism by sequencing on a population scale: "

Discovery and genotyping of genome structural polymorphism by sequencing on a population scale

Nature Genetics 43, 269 (2011). doi:10.1038/ng.768

Authors: Robert E Handsaker, Joshua M Korn, James Nemesh & Steven A McCarroll

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Long- and Short-Range Transcriptional Repressors Induce Distinct Chromatin States on Repressed Genes

Long- and Short-Range Transcriptional Repressors Induce Distinct Chromatin States on Repressed Genes: "Li M. Li, David N. Arnosti. Transcriptional repression is essential for establishing precise patterns of gene expression during development [1]. Repressors governing early Drosophila segmentation can be classified as ...."

Gene clustering pattern, promoter architecture, and gene expression stability in eukaryotic genomes [Evolution]

Gene clustering pattern, promoter architecture, and gene expression stability in eukaryotic genomes [Evolution]: "A balance between gene expression stability and evolvability is essential for the long-term maintenance of a living system. In this paper, we studied whether the genetic and epigenetic properties of the promoter affect gene expression variability. We hypothesized that upstream distance and orientation (head-to-head or head-to-tail) are important for the promoter architecture and gene expression variability. We found that in budding yeast genes with a short upstream distance tend to have low gene expression variability, and their promoter is flanked by strongly positioned nucleosomes and tends to have low nucleosome occupancy. These observations suggest that in vivo positioning of the flanking nucleosomes facilitates stable nucleosome depletion at the core promoter region and enhances gene expression stability. Head-to-head genes have, on average, lower gene expression variability, greater nucleosome depletion at the core promoter region, and more strongly positioned nucleosomes that flank the core promoter than do head-to-tail genes. These observations hold for diverse eukaryotes. In complex organisms such as mammals, only a small fraction of head-to-tail genes have retained a short upstream distance, probably because the promoter may not be flanked by a strongly positioned nucleosome on the upstream side."

Hox gene Ultrabithorax regulates distinct sets of target genes at successive stages of Drosophila haltere morphogenesis [Genetics]

Hox gene Ultrabithorax regulates distinct sets of target genes at successive stages of Drosophila haltere morphogenesis [Genetics]: "Hox genes encode highly conserved transcription factors that regionalize the animal body axis by controlling complex developmental processes. Although they are known to operate in multiple cell types and at different stages, we are still missing the batteries of genes targeted by any one Hox gene over the course of a single developmental process to achieve a particular cell and organ morphology. The transformation of wings into halteres by the Hox gene Ultrabithorax (Ubx) in Drosophila melanogaster presents an excellent model system to study the Hox control of transcriptional networks during successive stages of appendage morphogenesis and cell differentiation. We have used an inducible misexpression system to switch on Ubx in the wing epithelium at successive stages during metamorphosis—in the larva, prepupa, and pupa. We have then used extensive microarray expression profiling and quantitative RT-PCR to identify the primary transcriptional responses to Ubx. We find that Ubx targets range from regulatory genes like transcription factors and signaling components to terminal differentiation genes affecting a broad repertoire of cell behaviors and metabolic reactions. Ubx up- and down-regulates hundreds of downstream genes at each stage, mostly in a subtle manner. Strikingly, our analysis reveals that Ubx target genes are largely distinct at different stages of appendage morphogenesis, suggesting extensive interactions between Hox genes and hormone-controlled regulatory networks to orchestrate complex genetic programs during metamorphosis."

De-Novo Discovery of Differentially Abundant Transcription Factor Binding Sites Including Their Positional Preference

De-Novo Discovery of Differentially Abundant Transcription Factor Binding Sites Including Their Positional Preference: "Author Summary

Binding of transcription factors to promoters of genes, and subsequent enhancement or repression of transcription, is one of the main steps of transcriptional gene regulation. Direct or indirect wet-lab experiments allow the identification of approximate regions potentially bound or regulated by a transcription factor. Subsequently, de-novo motif discovery tools can be used for detecting the precise positions of binding sites. Many traditional tools focus on motifs over-represented in the target regions, which often turn out to be similarly over-represented in the entire genome. In contrast, several recent tools focus on differentially abundant motifs in target regions compared to a control set. As binding sites are often located at some preferred distance to the transcription start site, it is favorable to include this information into de-novo motif discovery. Here, we present Dispom a novel approach for learning differentially abundant motifs and their positional preferences simultaneously, which predicts binding sites with increased accuracy compared to many popular de-novo motif discovery tools. When applying Dispom to promoters of auxin-responsive genes of Arabidopsis thaliana, we find a binding motif slightly different from the canonical auxin-response element, which exhibits a strong positional preference and which is considerably more specific to auxin-responsive genes.

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Noncanonical Compensation of Zygotic X Transcription in Early Drosophila melanogaster Development Revealed through Single-Embryo RNA-Seq

Noncanonical Compensation of Zygotic X Transcription in Early Drosophila melanogaster Development Revealed through Single-Embryo RNA-Seq: "

Mmany genes from the X chromosome are expressed at the same level in female and male embryos during early Drosophila development, prior to the establishment of MSL-mediated dosage compensation, suggesting the existence of a novel mechanism.

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A unique chromatin signature uncovers early developmental enhancers in humans

A unique chromatin signature uncovers early developmental enhancers in humans: "

A unique chromatin signature uncovers early developmental enhancers in humans

Nature 470, 7333 (2011). doi:10.1038/nature09692

Authors: Alvaro Rada-Iglesias, Ruchi Bajpai, Tomek Swigut, Samantha A. Brugmann, Ryan A. Flynn & Joanna Wysocka

Cell-fate transitions involve the integration of genomic information encoded by regulatory elements, such as enhancers, with the cellular environment. However, identification of genomic sequences that control human embryonic development represents a formidable challenge. Here we show that in human embryonic stem cells (hESCs), unique chromatin signatures identify two distinct classes of genomic elements, both of which are marked by the presence of chromatin regulators p300 and BRG1, monomethylation of histone H3 at lysine 4 (H3K4me1), and low nucleosomal density. In addition, elements of the first class are distinguished by the acetylation of histone H3 at lysine 27 (H3K27ac), overlap with previously characterized hESC enhancers, and are located proximally to genes expressed in hESCs and the epiblast. In contrast, elements of the second class, which we term ‘poised enhancers’, are distinguished by the absence of H3K27ac, enrichment of histone H3 lysine 27 trimethylation (H3K27me3), and are linked to genes inactive in hESCs and instead are involved in orchestrating early steps in embryogenesis, such as gastrulation, mesoderm formation and neurulation. Consistent with the poised identity, during differentiation of hESCs to neuroepithelium, a neuroectoderm-specific subset of poised enhancers acquires a chromatin signature associated with active enhancers. When assayed in zebrafish embryos, poised enhancers are able to direct cell-type and stage-specific expression characteristic of their proximal developmental gene, even in the absence of sequence conservation in the fish genome. Our data demonstrate that early developmental enhancers are epigenetically pre-marked in hESCs and indicate an unappreciated role of H3K27me3 at distal regulatory elements. Moreover, the wealth of new regulatory sequences identified here provides an invaluable resource for studies and isolation of transient, rare cell populations representing early stages of human embryogenesis.

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Chromatin remodelling complex dosage modulates transcription factor function in heart development

Chromatin remodelling complex dosage modulates transcription factor function in heart development: "

Chromatin remodelling complex dosage modulates transcription factor function in heart development

Nature Communications 2, 187 (2011). doi:10.1038/ncomms1187

Authors: Jun K. Takeuchi, Xin Lou, Jeffrey M. Alexander, Hiroe Sugizaki, Paul Delgado-Olguín, Alisha K. Holloway, Alessandro D. Mori, John N. Wylie, Chantilly Munson, Yonghong Zhu, Yu-Qing Zhou, Ru-Fang Yeh, R. Mark Henkelman, Richard P. Harvey, Daniel Metzger, Pierre Chambon, Didier Y. R. Stainier, Katherine S. Pollard, Ian C. Scott & Benoit G. Bruneau

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Theoretical and empirical quality assessment of transcription factor-binding motifs

Theoretical and empirical quality assessment of transcription factor-binding motifs: "

Position-specific scoring matrices (PSSMs) are routinely used to predict transcription factor (TF)-binding sites in genome sequences. However, their reliability to predict novel binding sites can be far from optimum, due to the use of a small number of training sites or the inappropriate choice of parameters when building the matrix or when scanning sequences with it. Measures of matrix quality such as E-value and information content rely on theoretical models, and may fail in the context of full genome sequences. We propose a method, implemented in the program ‘matrix-quality’, that combines theoretical and empirical score distributions to assess reliability of PSSMs for predicting TF-binding sites. We applied ‘matrix-quality’ to estimate the predictive capacity of matrices for bacterial, yeast and mouse TFs. The evaluation of matrices from RegulonDB revealed some poorly predictive motifs, and allowed us to quantify the improvements obtained by applying multi-genome motif discovery. Interestingly, the method reveals differences between global and specific regulators. It also highlights the enrichment of binding sites in sequence sets obtained from high-throughput ChIP-chip (bacterial and yeast TFs), and ChIP–seq and experiments (mouse TFs). The method presented here has many applications, including: selecting reliable motifs before scanning sequences; improving motif collections in TFs databases; evaluating motifs discovered using high-throughput data sets.

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Minor change, major difference: divergent functions of highly conserved cis-regulatory elements subsequent to whole genome duplication events [RESEARCH REPORTS]

Minor change, major difference: divergent functions of highly conserved cis-regulatory elements subsequent to whole genome duplication events [RESEARCH REPORTS]: "Debbie K. Goode, Heather A. Callaway, Gustavo A. Cerda, Katharine E. Lewis, and Greg Elgar

Within the vertebrate lineage, a high proportion of duplicate genes have been retained after whole genome duplication (WGD) events. It has been proposed that many of these duplicate genes became indispensable because the ancestral gene function was divided between them. In addition, novel functions may have evolved, owing to changes in cis-regulatory elements. Functional analysis of the PAX2/5/8 gene subfamily appears to support at least the first part of this hypothesis. The collective role of these genes has been widely retained, but sub-functions have been differentially partitioned between the genes in different vertebrates. Conserved non-coding elements (CNEs) represent an interesting and readily identifiable class of putative cis-regulatory elements that have been conserved from fish to mammals, an evolutionary distance of 450 million years. Within the PAX2/5/8 gene subfamily, PAX2 is associated with the highest number of CNEs. An additional WGD experienced in the teleost lineage led to two copies of pax2, each of which retained a large proportion of these CNEs. Using a reporter gene assay in zebrafish embryos, we have exploited this rich collection of regulatory elements in order to determine whether duplicate CNEs have evolved different functions. Remarkably, we find that even highly conserved sequences exhibit more functional differences than similarities. We also discover that short flanking sequences can have a profound impact on CNE function. Therefore, if CNEs are to be used as candidate enhancers for transgenic studies or for multi-species comparative analyses, it is paramount that the CNEs are accurately delineated.

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Gene Expression Noise in Spatial Patterning: hunchback Promoter Structure Affects Noise Amplitude and Distribution in Drosophila Segmentation

Gene Expression Noise in Spatial Patterning: hunchback Promoter Structure Affects Noise Amplitude and Distribution in Drosophila Segmentation: "Author Summary

Noise is an intrinsic part of biochemical systems such as gene regulation networks. Noisy gene expression has been well documented in populations of single cells, and is likely a key mechanism in evolutionary change. But in developing embryos, cells within a tissue must overcome such variability in order to provide the uniformity required to coordinate multiple events. Reproducibility and determinacy of the spatial protein patterns preceding tissue differentiation is a critical aspect of development. In this study, we use anterior-posterior (AP) segmentation in the fruit fly (Drosophila) to understand how gene regulation dynamics control noise. One of the earliest AP patterning events is the anterior activation of the hunchback (hb) gene by the maternally-derived Bicoid (Bcd) protein gradient. This interaction has been very well characterized, providing the tools for us to develop a stochastic model of hb gene regulation to make predictions about expression noise, and to corroborate these experimentally. For hb, we show that self-regulation is a critical part of controlling noise, and the multiple Bcd binding sites in the hb promoter also enhance pattern reproducibility. To the degree that such features are shared by other genes, these noise-reducing mechanisms may be common to many pattern forming events.

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Structural Properties of the Caenorhabditis elegans Neuronal Network

Structural Properties of the Caenorhabditis elegans Neuronal Network: "Author Summary

Connectomics, the generation and analysis of neuronal connectivity data, stands to revolutionize neurobiology just as genomics has revolutionized molecular biology. Indeed, since neuronal networks are the physical substrates upon which neural functions are carried out, their structural properties are intertwined with the organization and logic of function. In this paper, we report a near-complete wiring diagram of the nematode Caenorhabditis elegans and present several analyses of its properties, finding many nonrandom features. We give novel visualizations and compute network statistics to enhance understanding of the reported data. We also use principled systems-theoretic methods to generate hypotheses on how biological function may arise from the reported neuronal network structure. The wiring diagram reported here can further be used to generate predictions about signal propagation in future perturbation, ablation, or artificial stimulation experiments.

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The Architecture of Gene Regulatory Variation across Multiple Human Tissues: The MuTHER Study

The Architecture of Gene Regulatory Variation across Multiple Human Tissues: The MuTHER Study: "Author Summary

Regulation of gene expression is a fundamental cellular process determining a large proportion of the phenotypic variance. Previous studies have identified genetic loci influencing gene expression levels (eQTLs), but the complexity of their tissue-specific properties has not yet been well-characterized. In this study, we perform cis-eQTL analysis in a unique matched co-twin design for three human tissues derived simultaneously from the same set of individuals. The study design allows validation of the substantial discoveries we make in each tissue. We explore in depth the tissue-dependent features of regulatory variants and estimate the proportions of shared and specific effects. We use continuous measures of eQTL sharing to circumvent the statistical power limitations of comparing direct overlap of eQTLs in multiple tissues. In this framework, we demonstrate that 30% of eQTLs are shared among tissues, while 29% are exclusively tissue-specific. Furthermore, we show that the fold change in expression between eQTL genotypic classes differs between tissues. Even among shared eQTLs, we report a substantial proportion (10%–20%) of significant tissue differences in magnitude of these effects. The complexities we highlight here are essential for understanding the impact of regulatory variants on complex traits.

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Quantitative Models of the Mechanisms That Control Genome-Wide Patterns of Transcription Factor Binding during Early Drosophila Development

Quantitative Models of the Mechanisms That Control Genome-Wide Patterns of Transcription Factor Binding during Early Drosophila Development: "Author Summary

During early stages of development, regulatory proteins bind DNA and control the expression of nearby genes, thereby driving spatial and temporal patterns of gene expression during development. But the biochemical forces that determine where these regulatory proteins bind are poorly understood. We gathered experimental data on the activities of several key regulators of early development of the fruit fly (Drosophila melanogaster) and developed a computational method to predict where and how strongly they will bind. We find that competition, cooperativity, and other interactions among individual regulatory proteins have a limited effect on their binding, while the global accessibility of DNA to protein binding has a significant impact on the binding of all factors. Our results suggest a practical method for predicting regulatory binding by combining experimental DNA accessibility assays with computational algorithms to determine where will binding occur among the accessible regions of the genome.

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Genome-wide transcription factor binding: beyond direct target regulation

Genome-wide transcription factor binding: beyond direct target regulation: "Kyle L. MacQuarrie, Abraham P. Fong, Randall H. Morse, Stephen J. Tapscott. The binding of transcription factors to specific DNA target sequences is the fundamental basis of gene regulatory networks. Chromatin immunoprecipitation combined with DNA tiling arrays or high-th...."

The transcriptional diversity of 25 Drosophila cell lines [RESOURCES]

The transcriptional diversity of 25 Drosophila cell lines [RESOURCES]: "

Drosophila melanogaster cell lines are important resources for cell biologists. Here, we catalog the expression of exons, genes, and unannotated transcriptional signals for 25 lines. Unannotated transcription is substantial (typically 19% of euchromatic signal). Conservatively, we identify 1405 novel transcribed regions; 684 of these appear to be new exons of neighboring, often distant, genes. Sixty-four percent of genes are expressed detectably in at least one line, but only 21% are detected in all lines. Each cell line expresses, on average, 5885 genes, including a common set of 3109. Expression levels vary over several orders of magnitude. Major signaling pathways are well represented: most differentiation pathways are 'off' and survival/growth pathways 'on.' Roughly 50% of the genes expressed by each line are not part of the common set, and these show considerable individuality. Thirty-one percent are expressed at a higher level in at least one cell line than in any single developmental stage, suggesting that each line is enriched for genes characteristic of small sets of cells. Most remarkable is that imaginal disc-derived lines can generally be assigned, on the basis of expression, to small territories within developing discs. These mappings reveal unexpected stability of even fine-grained spatial determination. No two cell lines show identical transcription factor expression. We conclude that each line has retained features of an individual founder cell superimposed on a common 'cell line' gene expression pattern.

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Diverse transcription factor binding features revealed by genome-wide ChIP-seq in C. elegans [RESEARCH]

Diverse transcription factor binding features revealed by genome-wide ChIP-seq in C. elegans [RESEARCH]: "

Regulation of gene expression by sequence-specific transcription factors is central to developmental programs and depends on the binding of transcription factors with target sites in the genome. To date, most such analyses in Caenorhabditis elegans have focused on the interactions between a single transcription factor with one or a few select target genes. As part of the modENCODE Consortium, we have used chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) to determine the genome-wide binding sites of 22 transcription factors (ALR-1, BLMP-1, CEH-14, CEH-30, EGL-27, EGL-5, ELT-3, EOR-1, GEI-11, HLH-1, LIN-11, LIN-13, LIN-15B, LIN-39, MAB-5, MDL-1, MEP-1, PES-1, PHA-4, PQM-1, SKN-1, and UNC-130) at diverse developmental stages. For each factor we determined candidate gene targets, both coding and non-coding. The typical binding sites of almost all factors are within a few hundred nucleotides of the transcript start site. Most factors target a mixture of coding and non-coding target genes, although one factor preferentially binds to non-coding RNA genes. We built a regulatory network among the 22 factors to determine their functional relationships to each other and found that some factors appear to act preferentially as regulators and others as target genes. Examination of the binding targets of three related HOX factors—LIN-39, MAB-5, and EGL-5—indicates that these factors regulate genes involved in cellular migration, neuronal function, and vulval differentiation, consistent with their known roles in these developmental processes. Ultimately, the comprehensive mapping of transcription factor binding sites will identify features of transcriptional networks that regulate C. elegans developmental processes.

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Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay

Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay: "Background:
Protein interactions control the regulatory networks underlying developmental processes. The understanding of developmental complexity will, therefore, require the characterization of protein interactions within their proper environment. The bimolecular fluorescence complementation (BiFC) technology offers this possibility as it enables the direct visualization of protein interactions in living cells. However, its potential has rarely been applied in embryos of animal model organisms and was only performed under transient protein expression levels.
Results:
Using a Hox protein partnership as a test case, we investigated the suitability of BiFC for the study of protein interactions in the living Drosophila embryo. Importantly, all BiFC parameters were established with constructs that were stably expressed under the control of endogenous promoters. Under these physiological conditions, we showed that BiFC is specific and sensitive enough to analyse dynamic protein interactions. We next used BiFC in a candidate interaction screen, which led to the identification of several Hox protein partners.
Conclusion:
Our results establish the general suitability of BiFC for revealing and studying protein interactions in their physiological context during the rapid course of Drosophila embryonic development."

Modularity of gene-regulatory networks revealed in sea-star development

Modularity of gene-regulatory networks revealed in sea-star development: "Evidence that conserved developmental gene-regulatory networks can change as a unit during deutersostome evolution emerges from a study published in BMC Biology. This shows that genes consistently expressed in anterior brain patterning in hemichordates and chordates are expressed in a similar spatial pattern in another deuterostome, an asteroid echinoderm (sea star), but in a completely different developmental context (the animal-vegetal axis). This observation has implications for hypotheses on the type of development present in the deuterostome common ancestor.See research article: http://www.biomedcentral.com/1741-7007/8/143/abstract"

Corrigendum: Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior

Corrigendum: Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior: "

Corrigendum: Two-photon calcium imaging from head-fixed Drosophila during optomotor walking behavior

Nature Methods 8, 184 (2011). doi:10.1038/nmeth0211-184b

Author: Johannes D Seelig, M Eugenia Chiappe, Gus K Lott, Anirban Dutta, Jason E Osborne, Michael B Reiser & Vivek Jayaraman

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A microfluidic array for large-scale ordering and orientation of embryos

A microfluidic array for large-scale ordering and orientation of embryos: "

A microfluidic array for large-scale ordering and orientation of embryos

Nature Methods 8, 171 (2011). doi:10.1038/nmeth.1548

Authors: Kwanghun Chung, Yoosik Kim, Jitendra S Kanodia, Emily Gong, Stanislav Y Shvartsman & Hang Lu

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One genome, two haplotypes

One genome, two haplotypes: "

One genome, two haplotypes

Nature Methods 8, 107 (2011). doi:10.1038/nmeth0211-107

Author: Nicole Rusk

Two approaches using either fosmid clones or a microfluidic device are used to tackle the challenge of a haplotype-resolved human genome.

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