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  • 1.
    Beloqui, Ana
    et al.
    Catholic Univ Louvain, Louvain Drug Res Inst, Dept Adv Drug Delivery & Biomat, Brussels, Belgium..
    Brayden, David J.
    Univ Coll Dublin, Sch Vet Med, Vet Biosci Sect, Dublin, Ireland.;Univ Coll Dublin, Conway Inst, Dublin, Ireland..
    Artursson, Per
    Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Preat, Veronique
    Catholic Univ Louvain, Louvain Drug Res Inst, Dept Adv Drug Delivery & Biomat, Brussels, Belgium..
    des Rieux, Anne
    Catholic Univ Louvain, Louvain Drug Res Inst, Dept Adv Drug Delivery & Biomat, Brussels, Belgium.;Catholic Univ Louvain, Inst Condensed Matter & Nanosci, Louvain Le Neuve, Belgium..
    A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation2017In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 12, no 7, p. 1387-1399Article in journal (Refereed)
    Abstract [en]

    The specialized microfold cells (M cells) in the follicle-associated epithelium (FAE) of intestinal Peyer's patches serve as antigen-sampling cells of the intestinal innate immune system. Unlike 'classical' enterocytes, they are able to translocate diverse particulates without digesting them. They act as pathways for microorganism invasion and mediate food tolerance by transcellular transport of intestinal microbiota and antigens. Their ability to transcytose intact particles can be used to develop oral drug delivery and oral immunization strategies. This protocol describes a reproducible and versatile human M-cell-like in vitro model. This model can be exploited to evaluate M-cell transport of microparticles and nanoparticles for protein, drug or vaccine delivery and to study bacterial adherence and translocation across M cells. The inverted in vitro M-cell model consists of three main steps. First, Caco-2 cells are seeded at the apical side of the inserts. Second, the inserts are inverted and B lymphocytes are seeded at the basolateral side of the inserts. Third, the conversion to M cells is assessed. Although various M-cell culture systems exist, this model provides several advantages over the rest: (i) it is based on coculture with well-established differentiated human cell lines; (ii) it is reproducible under the conditions described herein; (iii) it can be easily mastered; and (iv) it does not require the isolation of primary cells or the use of animals. The protocol requires skills in cell culture and microscopy analysis. The model is obtained after 3 weeks, and transport experiments across the differentiated model can be carried out over periods of up to 10 h.

  • 2. Haas, Brian J.
    et al.
    Papanicolaou, Alexie
    Yassour, Moran
    Grabherr, Manfred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Blood, Philip D.
    Bowden, Joshua
    Couger, Matthew Brian
    Eccles, David
    Li, Bo
    Lieber, Matthias
    MacManes, Matthew D.
    Ott, Michael
    Orvis, Joshua
    Pochet, Nathalie
    Strozzi, Francesco
    Weeks, Nathan
    Westerman, Rick
    William, Thomas
    Dewey, Colin N.
    Henschel, Robert
    Leduc, Richard D.
    Friedman, Nir
    Regev, Aviv
    De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis2013In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 8, no 8, p. 1494-1512Article in journal (Refereed)
    Abstract [en]

    De novo assembly of RNA-seq data enables researchers to study transcriptomes without the need for a genome sequence; this approach can be usefully applied, for instance, in research on 'non-model organisms' of ecological and evolutionary importance, cancer samples or the microbiome. In this protocol we describe the use of the Trinity platform for de novo transcriptome assembly from RNA-seq data in non-model organisms. We also present Trinity-supported companion utilities for downstream applications, including RSEM for transcript abundance estimation, R/Bioconductor packages for identifying differentially expressed transcripts across samples and approaches to identify protein-coding genes. In the procedure, we provide a workflow for genome-independent transcriptome analysis leveraging the Trinity platform. The software, documentation and demonstrations are freely available from http://trinityrnaseq.sourceforge.net. The run time of this protocol is highly dependent on the size and complexity of data to be analyzed. The example data set analyzed in the procedure detailed herein can be processed in less than 5 h.

  • 3.
    Lindhagen, Elin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    Nygren, Peter
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Oncology, Radiology and Clinical Immunology, Oncology.
    Larsson, Rolf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology.
    The fluorometric microculture cytotoxicity assay2008In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 3, no 8, p. 1364-1369Article in journal (Refereed)
    Abstract [en]

    The fluorometric microculture cytotoxicity assay (FMCA) is a nonclonogenic microplate-based cell viability assay used for measurement of the cytotoxic and/or cytostatic effect of different compounds in vitro. The assay is based on hydrolysis of the probe, fluorescein diacetate (FDA) by esterases in cells with intact plasma membranes. The assay is available as both a semiautomated 96-well plate setup and a 384-well plate version fully adaptable to robotics. Experimental plates are prepared with a small amount of drug solution and can be stored frozen. Cells are seeded on the plates and cell viability is evaluated after 72 h. The protocol described here is applicable both for cell lines and freshly prepared tumor cells from patients and is suitable both for screening in drug development and as a basis for a predictive test for individualization of anticancer drug therapy.

  • 4.
    Nong, Rachel Yuan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Wu, Di
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Yan, Junhong
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Hammond, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Gu, Gucci Jijuan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kamali-Moghaddam, Masood
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Landegren, Ulf
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Darmanis, Spyros
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Solid-phase proximity ligation assays for individual or parallel protein analyses with readout via real-time PCR or sequencing2013In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 8, no 6, p. 1234-1248Article in journal (Refereed)
    Abstract [en]

    Solid-phase proximity ligation assays share properties with the classical sandwich immunoassays for protein detection. The proteins captured via antibodies on solid supports are, however, detected not by single antibodies with detectable functions, but by pairs of antibodies with attached DNA strands. Upon recognition by these sets of three antibodies, pairs of DNA strands brought in proximity are joined by ligation. The ligated reporter DNA strands are then detected via methods such as real-time PCR or next-generation sequencing (NGS). We describe how to construct assays that can offer improved detection specificity by virtue of recognition by three antibodies, as well as enhanced sensitivity owing to reduced background and amplified detection. Finally, we also illustrate how the assays can be applied for parallel detection of proteins, taking advantage of the oligonucleotide ligation step to avoid background problems that might arise with multiplexing. The protocol for the singleplex solid-phase proximity ligation assay takes similar to 5 h. The multiplex version of the assay takes 7-8 h depending on whether quantitative PCR (qPCR) or sequencing is used as the readout. The time for the sequencing-based protocol includes the library preparation but not the actual sequencing, as times may vary based on the choice of sequencing platform.

  • 5.
    Sanchez, Sophie
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology.
    Fernandez, Vincent
    Pierce, Stephanie E.
    Tafforeau, Paul
    Homogenization of sample absorption for the imaging of large and dense fossils with synchrotron microtomography2013In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 8, no 9, p. 1708-1717Article in journal (Refereed)
    Abstract [en]

    Propagation phase-contrast synchrotron radiation microtomography (PPC-SR mu CT) has proved to be very successful for examining fossils. Because fossils range widely in taphonomic preservation, size, shape and density, X-ray computed tomography protocols are constantly being developed and refined. Here we present a 1-h procedure that combines a filtered high-energy polychromatic beam with long-distance PPC-SR mu CT (sample to detector: 4-16 m) and an attenuation protocol normalizing the absorption profile (tested on 13-cm-thick and 5.242 g cm(-3) locally dense samples but applicable to 20-cm-thick samples). This approach provides high-quality imaging results, which show marked improvement relative to results from images obtained without the attenuation protocol in apparent transmission, contrast and signal-to-noise ratio. The attenuation protocol involves immersing samples in a tube filled with aluminum or glass balls in association with a U-shaped aluminum profiler. This technique therefore provides access to a larger dynamic range of the detector used for tomographic reconstruction. This protocol homogenizes beam-hardening artifacts, thereby rendering it effective for use with conventional mu CT scanners.

  • 6. Schnorrer, Frank
    et al.
    Ahlford, Annika
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Chen, Doris
    Milani, Lili
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Syvänen, Ann-Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular Medicine.
    Positional cloning by fast-track SNP-mapping in Drosophila melanogaster2008In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 3, no 11, p. 1751-1765Article in journal (Refereed)
    Abstract [en]

    Positional cloning of chemically induced mutations is the rate-limiting step in forward genetic screens in Drosophila. Single-nucleotide polymorphisms (SNPs) are useful markers to locate a mutated region in the genome. Here, we provide a protocol for high-throughput, high-resolution SNP mapping that enables rapid and cost-effective positional cloning in Drosophila. In stage 1 of the protocol, we use highly multiplexed tag-array mini-sequencing assays to map mutations to an interval of 1-2 Mb. In these assays, SNPs are genotyped by primer extension using fluorescently labeled dideoxy-nucleotides. Fluorescent primers are captured and detected on a microarray. In stage 2, we selectively isolate recombinants within the identified 1-2 Mb interval for fine mapping of mutations to about 50 kb. We have previously demonstrated the applicability of this protocol by mapping 14 muscle morphogenesis mutants within 4 months, which represents a significant acceleration compared with other commonly used mapping strategies that may take years.

  • 7.
    Varshney, Gaurav K.
    et al.
    NHGRI, Dev Genom Sect, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA.;Oklahoma Med Res Fdn, Funct & Chem Genom Program, 825 NE 13th St, Oklahoma City, OK 73104 USA..
    Carrington, Blake
    NHGRI, Zebrafish Core, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
    Pei, Wuhong
    NHGRI, Dev Genom Sect, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
    Bishop, Kevin
    NHGRI, Zebrafish Core, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
    Chen, Zelin
    NHGRI, Dev Genom Sect, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
    Fan, Chunxin
    Shanghai Ocean Univ, Minist Educ, Key Lab Explorat & Utilizat Aquat Genet Resource, Shanghai, Peoples R China..
    Xu, Lisha
    NHGRI, Dev Genom Sect, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
    Jones, Marypat
    NHGRI, Canc Genet & Comparat Genom Branch, NIH, Bethesda, MD 20892 USA..
    LaFave, Matthew C.
    NHGRI, Dev Genom Sect, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA.;Synthet Genom, San Diego, CA USA..
    Ledin, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Organismal Biology, Evolution and Developmental Biology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Sood, Raman
    NHGRI, Zebrafish Core, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
    Burgess, Shawn M.
    NHGRI, Dev Genom Sect, Translat & Funct Genom Branch, NIH, Bethesda, MD 20892 USA..
    A high-throughput functional genomics workflow based on CRISPR/Cas9-mediated targeted mutagenesis in zebrafish2016In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 11, no 12, p. 2357-2375Article in journal (Refereed)
    Abstract [en]

    The zebrafish is a popular model organism for studying development and disease, and genetically modified zebrafish provide an essential tool for functional genomic studies. Numerous publications have demonstrated the efficacy of gene targeting in zebrafish using CRISPR/Cas9, and they have included descriptions of a variety of tools and methods for guide RNA synthesis and mutant identification. However, most of the published techniques are not readily scalable to increase throughput. We recently described a CRISPR/Cas9-based high-throughput mutagenesis and phenotyping pipeline in zebrafish. Here, we present a complete workflow for this pipeline, including target selection; cloning-free single-guide RNA (sgRNA) synthesis; microinjection; validation of the target-specific activity of the sgRNAs; founder screening to identify germline-transmitting mutations by fluorescence PCR; determination of the exact lesion by Sanger or next-generation sequencing (including software for analysis); and genotyping in the F-1 or subsequent generations. Using these methods, sgRNAs can be evaluated in 3 d, zebrafish germline-transmitting mutations can be identified within 3 months and stable lines can be established within 6 months. Realistically, two researchers can target tens to hundreds of genes per year using this protocol.

  • 8.
    Weibrecht, Irene
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Lundin, Elin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Kiflemariam, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Mignardi, Marco
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Grundberg, Ida
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Larsson, Chatarina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Genomics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Koos, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Nilsson, Mats
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Söderberg, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.
    In situ detection of individual mRNA molecules and protein complexes or post-translational modifications using padlock probes combined with the in situ proximity ligation assay2013In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 8, no 2, p. 355-372Article in journal (Refereed)
    Abstract [en]

    Analysis at the single-cell level is essential for the understanding of cellular responses in heterogeneous cell populations, but it has been difficult to perform because of the strict requirements put on detection methods with regard to selectivity and sensitivity (i.e., owing to the cross-reactivity of probes and limited signal amplification). Here we describe a 1.5-d protocol for enumerating and genotyping mRNA molecules in situ while simultaneously obtaining information on protein interactions or post-translational modifications; this is achieved by combining padlock probes with in situ proximity ligation assays (in situ PLA). In addition, we provide an example of how to design padlock probes and how to optimize staining conditions for fixed cells and tissue sections. Both padlock probes and in situ PLA provide the ability to directly visualize single molecules by standard microscopy in fixed cells or tissue sections, and these methods may thus be valuable for both research and diagnostic purposes.

  • 9. Winkler, Thomas W.
    et al.
    Day, Felix R.
    Croteau-Chonka, Damien C.
    Wood, Andrew R.
    Locke, Adam E.
    Maegi, Reedik
    Ferreira, Teresa
    Fall, Tove
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular epidemiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Graff, Mariaelisa
    Justice, Anne E.
    Luan, Jian'an
    Gustafsson, Stefan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Molecular epidemiology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Randall, Joshua C.
    Vedantam, Sailaja
    Workalemahu, Tsegaselassie
    Kilpelainen, Tuomas O.
    Scherag, Andre
    Esko, Tonu
    Kutalik, Zoltan
    Heid, Iris M.
    Loos, Ruth J. F.
    Quality control and conduct of genome-wide association meta-analyses2014In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 9, no 5, p. 1192-1212Article in journal (Refereed)
    Abstract [en]

    Rigorous organization and quality control (QC) are necessary to facilitate successful genome-wide association meta-analyses (GWAMAs) of statistics aggregated across multiple genome-wide association studies. This protocol provides guidelines for (i) organizational aspects of GWAMAs, and for (ii) QC at the study file level, the meta- level across studies and the meta-analysis output level. Real-world examples highlight issues experienced and solutions developed by the GIANT Consortium that has conducted meta-analyses including data from 125 studies comprising more than 330,000 individuals. We provide a general protocol for conducting GWAMAs and carrying out QC to minimize errors and to guarantee maximum use of the data. We also include details for the use of a powerful and flexible software package called EasyQC. Precise timings will be greatly influenced by consortium size. For consortia of comparable size to the GIANT Consortium, this protocol takes a minimum of about 10 months to complete.

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