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Authors: Fabio Grati, Martina Scanu, Luca Bolognini
Summary: This protocol refers to a study having the aim to identify and test at field artificial structures suitable as collectors for common cuttlefish Sepia officinalis eggs in wild condition.During the same study, three different protocols were tested and compared. This one refers to the named 'seagrass' collector protocol, due to their similarity to the natural seagrass meadaw.This work was supported by the project “Protection, improvement and integrated management of the sea environment and of cross-border natural resources – ECOSEA”, funded by EU Adriatic IPA Cross-border Cooperation 2007-2013 (2°ord./0236/0).
Proper citation: Fabio Grati, Martina Scanu, Luca Bolognini 2018. "Seagrass" eggs collector for common cuttlefish . protocols.io dx.doi.org/10.17504/protocols.io.shheb36 Copy
Authors: Alejandro Morales Suarez, Susan Rutherford
Summary: This extraction protocol follows the DArT DNA Extraction protocol but contains modifications (including increasing the concentration of PVP to 4 %
Proper citation: Alejandro Morales Suarez, Susan Rutherford 2018. gDNA Extraction of Eucalypts pauciflora for full genome sequencing. protocols.io dx.doi.org/10.17504/protocols.io.j7ecrje Copy
Authors: Qi Y
Proper citation: Qi Y 2019. Sample Preparation for Western Blot. protocols.io dx.doi.org/10.17504/protocols.io.z7xf9pn Copy
Authors: Mariella Ferrante, Rossella Annunziata, Monia Teresa Russo, Francesco Manfellotto
Summary: Axenic cultures protocolAxenicity of cultures is obtained by multi-antibiotic treatment: by adding antibiotics directly to the medium in which the strain will be inoculated. After a variable amount of days we refresh the coltures with new medium.
Proper citation: Mariella Ferrante, Rossella Annunziata, Monia Teresa Russo, Francesco Manfellotto 2020. Axenic Diatoms cultures protocol. protocols.io dx.doi.org/10.17504/protocols.io.bgudjws6 Copy
Authors: Dr. Steven Wilhelm, Samantha Coy
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), Samantha R Coy's Protocols
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.
Proper citation: Dr. Steven Wilhelm, Samantha Coy 2017. Using the Biomate 5 Spectrophotometer. protocols.io dx.doi.org/10.17504/protocols.io.iktccwn Copy
Authors: Megan Huchko, Elizabeth Bukusi, Craig R. Cohen
Proper citation: Megan Huchko, Elizabeth Bukusi, Craig R. Cohen 2019. Evaluating a Community-Driven Cervical Cancer Prevention program in Western Kenya. protocols.io dx.doi.org/10.17504/protocols.io.6s5heg6 Copy
Authors: Anna Racioppi, Tamara Sheinbaum, Georgina M. Gross, Sergi Ballespí, Thomas R. Kwapil, Neus Barrantes-Vidal
Summary: The present study forms part of an ongoing longitudinal project examining risks and resilience for schizophrenia-spectrum disorders in a non-clinical ascertained sample of college students. The main objective of the study was examine the validity of psychometrically assessed positive and negative schizotypy at a three-year follow-up assessment (Time 3; T3) of the sample initially reported in the cross-sectional study of Barrantes-Vidal et al. (2013). Participants were recruited from the undergraduate population enrolled in Psychology courses at the Universitat Autònoma de Barcelona (UAB). A group of 589 unselected students completed a mass screening assessment of self-reported measures at Time 1 (T1). Participants were administered the Wisconsin Schizotypy Scales (WSS) intermixed with an infrequency scale, the positive symptoms subscale of the Community Assessment of Psychic Experiences (CAPE), and the suspiciousness subscale of the Schizotypal Personality Questionnaire (SPQ). Usable screening data were obtained from 547 students (mean age = 20.6; SD = 4.1; 86% female, 42 were excluded from the final study due to invalid protocols). Two interview and self-report measures assessments were conducted after T1. The Time 2 (T2) and Time 3 (T3) interviews were made by psychologists and advanced graduate students in clinical psychology. All interviewers were extensively trained and were unaware of participants' scores on the T1 and T2 measures. Of the 547 students who completed T1 assessment, a subset of 339 was invited to participate at T2 assessment with the aim of assessing 200 individuals. In order to ensure adequate inclusion of participants reporting high levels of schizotypy, the students group included 189 who had elevated scores (standard scores based upon sample norms of at least 1.0) on the positive or negative schizotypy factors derived from the WSS, or the SPQ-suspiciousness, or the CAPE, and 150 randomly selected participants who had standard scores below 1.0 on each of these measures.A total of 214 students (mean age = 21.4 years; SD = 2.4; 78% female) completed T2 assessment which took place an average of 1.7 years (SD= 0.2 years, range 1.4 to 2.2 years) after T1. This sample included 123 participants with at least one schizotypy screening score above 1.0 and 91 with standard scores below 1.0. The T3 assessment took place an average of 1.4 years (SD= 0.3 years, range 0.9 to 2.1 years) after T2, and of 3.1 years (SD= 0.3 years, range 2.6 to 3.6 years) after T1. At T3, due to financial limitation we targeted a subset of 134 students (93 with high schizotypy and 41 with standard scores below 1.0). Of these, 103 (77%) participants (mean age= 23.06; SD=2.6; 37.9% male) were interviewed at T3. The sample included 75 of 93 (82%) participants with elevated schizotypy scores and 28 of 43 (65%) with standard scores below 1.0. There were no significant differences on positive or negative schizotypy scores between the participants assessed at T3 and the non-followed participants. This study was conducted according to guidelines of the University Ethics Committee of the UAB and participants provided informed consent at each assessment.
Proper citation: Anna Racioppi, Tamara Sheinbaum, Georgina M. Gross, Sergi Ballespí, Thomas R. Kwapil, Neus Barrantes-Vidal 2018. Protocol of the Psychometric High-Risk Strategy Project for Examining Risk and Resilience Trajectories across the Psychosis Continuum. protocols.io dx.doi.org/10.17504/protocols.io.ubieske Copy
Authors: Kenneth Schackart
Group: Yoon Lab
Summary: How to prepare and run gel electrophoresis using agarose gel for the verification of nucleic acid amplification products.
Proper citation: Kenneth Schackart 2019. Gel Electrophoresis. protocols.io dx.doi.org/10.17504/protocols.io.yvxfw7n Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is for isolating monoclonal cell population by limiting dilution. To see the full abstract and additional resources, please visit the Addgene protocol page.Sample Data1lentiCas9-Blast was a gift from Feng Zhang (Addgene plasmid #52962) and is described in Improved vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods. 2014 Aug;11(8):783-4.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Isolating a Monoclonal Cell Population by Limiting Dilution. protocols.io dx.doi.org/10.17504/protocols.io.4xvgxn6 Copy
Authors: Nikita Joshi, Alexander Misharin
Group: Human Cell Atlas Method Development Community
Summary: We aimed to develop a protocol for isolation of the single nuclei from the archival frozen human lung tissue suitable for single cell RNA-seq using standard 10x Genomics chemistry. The protocol utilizes standard nuclei isolation buffer (Nuclei EZ buffer) supplemented with RNase inhibitor and a protease inhibitor. For the homogenization step, we elected to use C Tube and GentleMACS tissue dissociation as a way to standardize the homogenization procedure. To maintain nuclei integrity we skipped washing steps and instead use large volumes of washing buffer and proceed to FACSorting immediately after lysis for nuclei purification.This protocol produces a good yield of nuclei and diverse libraries, with multiple cell types being detected. While the number of detected genes and UMI per nuclei is lower than for single cell RNAseq, it is sufficient for identification of the specific cell types, including rare populations. Moreover, snRNAseq allowed resolution of the cell types that are intimately integrated into the lung matrix and otherwise hard to resolve: fibroblasts and type 1 alveolar epithelial cells.We thank Oni Basu (UChicago), Luciano Martelotto (Monash University), Nicole Abreu (10x Genomics) and Sharmila Chatterjee (10x Genomics) for their advice.
Proper citation: Nikita Joshi, Alexander Misharin 2019. Single-nucleus isolation from frozen human lung tissue for single-nucleus RNA-seq. protocols.io dx.doi.org/10.17504/protocols.io.zu8f6zw Copy
Authors: Diana Wider, Didier Picard
Summary: This is a novel dual reporter assay for transfected tissue culture cells. It is based on the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations and should be an attractive alternative to the currently available commercial kits for dual reporter protein assays.
Proper citation: Diana Wider, Didier Picard 2017. Secreted dual reporter assay with Gaussia luciferase and mCherry. protocols.io dx.doi.org/10.17504/protocols.io.kbwcspe Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the process for diaminobenzidine (DAB) detection of biocytin filled cells. This protocol is optimized for use with brain slices cut at 350 µm thick, in which cells are first filled with biocytin (i.e., post-electrophysiological recording), fixed in 4% PFA/2.5% glutaraldehyde, and transferred to PBS until ready to stain.Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Proper citation: Allen Institute for Brain Science 2020. DAB Detection of Biocytin Labeled Tissue. protocols.io dx.doi.org/10.17504/protocols.io.bdpji5kn Copy
Authors: David Eccles
Group: Coronavirus Method Development Community
Summary: This is a fast "gunk to graph" protocol for analysing viral RNA from nasopharyngeal swabs. The approach involves swab lysis and inactivation at the point of sampling, uses a cellulose binding / wash protocol to reduce extraction cost, incorporates sample-specific barcodes during first-strand synthesis, nanopore rapid-attachment primers during PCR amplification, and nanopore sequencing with parallel RAMPART analysis for fast assembly and phylogenetics.Note: this is a one-step RT-PCR reaction protocol. For an alternative method that allows more multiplexing flexibility, see the protocol that this has been forked from:https://dx.doi.org/10.17504/protocols.io.bd3yi8pw
Proper citation: David Eccles 2020. Viral Sequencing, from Gunk to Graph (One-Step four-primer PCR alternative). protocols.io dx.doi.org/10.17504/protocols.io.bd5ni85e Copy
Authors: Lutz Berwanger
Group: Axmann Lab, CyanoWorld
Summary: This protocol can be used to purifiy DNA-dependent RNA polymerase from Synechococcus elongatus PCC 7942 by heparin and ion-exchange chromatography.
Proper citation: Lutz Berwanger 2020. Purification of DNA-dependent RNA Polymerase from Synechococcus elongatus PCC 7942. protocols.io dx.doi.org/10.17504/protocols.io.bbmfik3n Copy
Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. They have both paracrine and autocrine function. Interleukins are also used in animal studies to investigate aspect related to clinical medicine.T cells and stem cells make IL-3. It functions as a multilineage colony-stimulating factor [1]Reference1. Justiz Vaillant AA, Qurie A. Interleukin. In:StatPearls. Treasure Island (FL): StatPearls Publishing; June 12, 2019.
Proper citation: Angel Justiz-Vaillant 2020. ELISA for quantification of IL-3 in human serum.. protocols.io dx.doi.org/10.17504/protocols.io.bj23kqgn Copy
Authors: Marine Vautier, Valentin Vasselon, Cecile Chardon, Frédéric Rimet, Agnès Bouchez, Isabelle Domaizon
Group: EcoALpsWater
Summary: This protocol is part of the DNA workflow applied in the Eco-AlpsWater Project.The methodological step described here is the extraction of DNA, this is a critical step for obtaining relevant results since molecular inventories might be influenced by the DNA extraction method used.The choice of the methodology for biofilms DNA extraction is based on previous studies and in particular on the work done by Vasselon et al. (2017) where the authors have tested 5 DNA extraction methods combining various types of cell lysis and DNA purification to extract DNA (from pure diatom cultures and biofilms samples from streams and lakes).For the Eco-AlpsWater project, after being sampled in lakes or rivers, biofilms are stored in 50 mL tubes in ethanol at 4°C, and for a maximum of 3 months before DNA extraction (the extraction should preferably be done in the month following the sampling).The DNA extraction protocol presented below has been used in several recent studies (e.g. Vasselon et al 2017ab, 2018) focussed on the application of diatoms metabarcoding; this extraction is based on a protocol adapted from the NucleoSpin® Soil kit (MACHEREY-NAGEL) with specific modifications for biofilm DNA extraction.
Proper citation: Marine Vautier, Valentin Vasselon, Cecile Chardon, Frédéric Rimet, Agnès Bouchez, Isabelle Domaizon 2020. DNA extraction from environmental biofilm using the NucleoSpin® Soil kit (MACHEREY-NAGEL) . protocols.io dx.doi.org/10.17504/protocols.io.bd52i88e Copy
Authors: Jason Kim
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary:In-cage free running wheel is used to voluntarily induce exercise in awake mice. Exercise affects insulin sensitivity and glucose metabolism.
Proper citation: Jason Kim 2019. U Mass - Exercise model. protocols.io dx.doi.org/10.17504/protocols.io.xwyfpfw Copy
Authors: lissa coffeey
Group: WordPress Security Protocols
Proper citation: lissa coffeey 2020. Cybersecurity Protocols & Resilience Post COVID - Lessons Learnt. protocols.io dx.doi.org/10.17504/protocols.io.bidxka7n Copy
Authors: Sanjay Gautam, Rajendra KC, Kelvin WC Leong, Micheál Mac Aogáin, Ronan F. O’Toole
Summary: Bacterial whole genome sequencing (WGS) is becoming a widely-used technique in research, clinical diagnostic, and public health laboratories. It enables high resolution characterization of bacterial pathogens in terms of properties that include antibiotic resistance, molecular epidemiology, and virulence. The introduction of next-generation sequencing instrumentation has made WGS attainable in terms of costs. However, the lack of a beginner’s protocol for WGS still represents a barrier to its adoption in some settings. Here, we present detailed step-by-step methods for obtaining WGS data from a range of different bacteria (Gram-positive, Gram-negative, and acid-fast) using the Illumina platform. Modifications have been performed with respect to DNA extraction and library normalization to maximize the output from the laboratory consumables invested. The protocol represents a simplified and reproducible method for producing high quality sequencing data. The key advantages of this protocol include simplicity of the protocol for users with no prior genome sequencing experience and reproducibility of the protocol across a wide range of bacteria.
Proper citation: Sanjay Gautam, Rajendra KC, Kelvin WC Leong, Micheál Mac Aogáin, Ronan F. O’Toole 2019. A step-by-step beginner’s protocol for whole genome sequencing of human bacterial pathogens. protocols.io dx.doi.org/10.17504/protocols.io.4xxgxpn Copy
Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: This protocols is to be performed with the Quick Ligation Reaction Buffer. Please see the NEB website for more information.
Proper citation: New England Biolabs 2014. Quick Ligation Protocol (M2200). protocols.io dx.doi.org/10.17504/protocols.io.cdgs3v Copy
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