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Authors: Yulin Wang, Zha Sang, Shaohang Xu, Qijun Xu, Xingquan Zeng, Dunzhu Jabu, Hongjun Yuan
Summary: This protocol describes the complete procedures for performing data‐independent acquisition‐based analysis from Tibetan hulless barley samples under osmotic stress.
Proper citation: Yulin Wang, Zha Sang, Shaohang Xu, Qijun Xu, Xingquan Zeng, Dunzhu Jabu, Hongjun Yuan 2020. Proteomic protocol for Tibetan hulless barley under osmotic stress. protocols.io dx.doi.org/10.17504/protocols.io.bbusinwe Copy
Authors: Li Deng
Group: VERVE Net, Sullivan Lab
Summary: Case 2: Fix samples. Use this protocol when long term storage of slide is required.For when you can count your samples in a few days, see Case 1.
Proper citation: Li Deng 2016. SYBR Gold Staining for Viral Enumeration (Case 2). protocols.io dx.doi.org/10.17504/protocols.io.c7fzjm Copy
Authors: Sam Li
Group: BioLegend
Proper citation: Sam Li 2019. Immunohistochemistry Protocol for Keratin Antibodies. protocols.io dx.doi.org/10.17504/protocols.io.95ph85n Copy
Authors: Ian Mackay, Judy Northill, Alyssa Pyke
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol was designed and developed at this laboratory and incorporates a previously published oligoprobe (see below).
The protocol specifically aims to amplify DENV02 viruses and not other dengue viruses. The assay targets the capsid region and is designed as a qualitative test for investigating suspected human cases of DENV-2 infections.
This assay has been superseded by the Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assay.
Proper citation: Ian Mackay, Judy Northill, Alyssa Pyke 2018. Dengue virus type 2 (DENV-2) capsid-Thai TaqMan assay (no longer in use; see Guidelines). protocols.io dx.doi.org/10.17504/protocols.io.q4ydyxw Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol goes through AAV purification by Iodixanol gradient ultracentrifugation. To see the full abstract and additional resources, visit the Addgene protocol page.Sample Data
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. AAV Purification by Iodixanol Gradient Ultracentrifugation. protocols.io dx.doi.org/10.17504/protocols.io.47jgzkn Copy
Authors: Marie Anne Balanant
Summary: This protocol is used to label red blood cells (RBC) for confocal imaging. Fluorescent beads are used to calibrate voxel size on the confocal microscope. The imaging protocol was optimised to obtain high quality data, by mounting the cells in a high refractive index and optimising acquisition parameters.Spherical aberration results in a decrease in image intensity when the imaging plan gets further away from the coverslip. It is caused by difference in refractive indices between the different components present over the beam pathway. In order to reduce spherical aberration, the cells are fixed in glutaraldehyde after staining so they can be resuspended in a medium with a high glycerol content and maintain their shape. Glycerol increases the reflective index of the medium, bringing it closer to the reflective indices of the coverslip and the immersion oil.Acquisition parameters are first chosen using the system optimisation function, then adjusted. The z-axis step size is chosen as close as possible to the lateral resolution to avoid under- or over-sampling. The pinhole is reduced to 50 μm to increase resolution and reduce out-of-focus light. A 2-step line averaging is selected during scanning to remove noise.Data is analysed and quantitative volume and surface area values were extracted using a home-written matlab code (not included in this protocol).
Proper citation: Marie Anne Balanant 2019. Preparation of human Red Blood Cells for confocal imaging. protocols.io dx.doi.org/10.17504/protocols.io.yjyfupw Copy
Authors: Vivian Liu
Proper citation: Vivian Liu 2015. Crude Membrane protein extraction from tissues. protocols.io dx.doi.org/10.17504/protocols.io.djt4nm Copy
Authors: Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood
Group: Human BioMolecular Atlas Program (HuBMAP) Method Development Community, GE Research
Summary: This protocol describes the process of validating antibodies (primary/secondary, direct conjugates, and zenon labelled) as per the Cell DIVE™ technology. It also describes the process for determining any antigen effects from the dye inactivation process.
Proper citation: Liz McDonough, Chrystal Chadwick, Fiona Ginty, Christine Surrette, Anup Sood 2020. Cell DIVE™ Platform | Antibody Characterization for Multiplexing. protocols.io dx.doi.org/10.17504/protocols.io.bpyxmpxn Copy
Authors: Jackie Gnepp, Joshua Klayman, Ian O. Williamson, Sema Barlas
Proper citation: Jackie Gnepp, Joshua Klayman, Ian O. Williamson, Sema Barlas 2020. The future of feedback: Motivating performance improvement through future-focused feedback --Study 2. protocols.io dx.doi.org/10.17504/protocols.io.bftsjnne Copy
Authors: Amin Mohamed, Bradley Evans, Antonio Reverter, James Kijas
Group: Salmon Multiomics
Summary: Background:Atlantic salmon farming promotes growth in conditions which mean animals may complete sexual development at weights below harvest size leading to a reduction in productivity. This prompted us to investigate the biological mechanisms that control the timing of sexual maturation. We performed a time course experiment, whereby animals were manipulated with photoperiod before tissues were collected across the time window when animals commence sexual development. We performed whole genome bisulfite sequencing (WGBS9 of three salmon tissues (pituitary, ovary and liver) at both the beginning and end of the experiment (T1 and T4), to take a first look at the patterns of DNA methylation and examine how they change in response to the onset of an important life history trait. Results:Comparison across timepoints revealed 6,373 differentially methylated regions (DMRs), of which approximately 50% were located within genes (DMGs). The ovary underwent the most profound remodelling, with a strong bias towards increased methylation levels (hyper-methylation) specially at gene bodies (gene body methylation). The majority of differentially methylated genes (DMGs) in ovary were hypermethylated (n=1165; 74%) and significantly enriched for three biological process (GO-BP), one cellular component (GO-CC) and 24 molecular function (GO-MF) terms including those with maturation-related functions such assemaphorin/glutamate receptor activity. We also performed deep transcriptomic profiling (RNA-seq) of the same tissues to explore the relationship between methylation changes and gene expression. Weak correlation was observed considering all available genes, suggesting methylation may not be the key epigenomic regulator of global expression in the context of our experiment. However, the identification of significant transcriptional and methylation changes allowed us to explore the dynamic between these two processes by assessing the overlap of genes declared as both DEG and DMG. The overlap was low and non-significant for liver (38 / 616 or 6% of DMGs were also DEGs) and pituitary (11 / 762 or 1.4% of DMGs were DEGs). Strikingly, 195 or 14% of ovary DMGs (195 / 1357) were also differentially expressed, a number that exceeded random expectation in 83.8% of 1000 permutations tests. This suggests changes in methylation status may directly control gene expression in this subset of genes. If true, we would expect to see correspondence between the directionality of the expression and methylation changes. This appeared to be the case, as 82% of upregulated genes (148 / 179; Binomial P-value = 8.727E-20) were hyper-methylated at T4 relative to T1, matching the classical expectation of gene body methylation mediated control of gene expression (Neri et al 2017, Arechederra et al 2018). The 148 genes were enriched for 3 GO-CC terms related to chromatin remodelling complexes (SWI/SNF and nBAF) and the associated genes displayed coordinated expression and methylation status.Conclusion and implications:Taken together, the results confirmed that while methylation alone does not control genome-wide patterns of gene expression, it plays a key role upregulating a defined set of genes during the maturation process. Co-analysis of transcriptome and DNA methylome in ovary suggests chromatin remodelling genes play a role in the commitment of animals to the sexual maturation pathway. These results also open the way for the identification of functional variants that can be used in advanced breeding approaches to boost productivity in Atlantic salmon farming.References:Neri, F. et al. Intragenic DNA methylation prevents spurious transcription initiation.Nature 543, 72–77 (2017).Arechederra, M. et al. Hypermethylation of gene body CpG islands predicts high dosage of functional oncogenes in liver cancer. Nat. Commun. 9, 3164 (2018).
Proper citation: Amin Mohamed, Bradley Evans, Antonio Reverter, James Kijas 2020. Multitissue DNA methylome profiling during onset of salmon maturation. protocols.io dx.doi.org/10.17504/protocols.io.bncrmav6 Copy
Authors: Kelsey Miller
Group: BioLegend
Summary: The HLA class I ELISA is an enzyme immunoassay based on the detection of β2-microglobulin subunit of HLA class I complexes, after capturing the complex through the conjugated biotin. To this end, biotinylated HLA class I complex is first captured in streptavidin coated microtiter wells. Subsequently, HRP-conjugated anti-human β2-microglobulin is added to detect intact HLA class I complexes. Only intact HLA class I complexes are recognized. Peptides with high affinity binding will be clearly detected by this ELISA technique, while peptides with a moderate to low binding affinity for HLA class I provide a moderate to non-detectable signal. This protocol is designed to evaluate the efficiency of peptide exchange when using the Flex-T™ system.
Proper citation: Kelsey Miller 2017. Flex-T™ HLA Class I ELISA Protocol. protocols.io dx.doi.org/10.17504/protocols.io.mcsc2we Copy
Authors: John Palmer
Summary: This is a protocol for sampling people in order to understand patterns of mobility and activity-space segregation at the scale of city-days. The goal is to obtain a representative sample of the space-time paths that exist in a given city. Sampling points and times are choosen and then used to recruit participants. If participants consent to be part of the study, they (1) respond anonymously to a survey about their activity-spaces, and/or (2) use a mobile phone application to track themselves during a 1-week period and share their anonymized trajectory information with the research team. This protocol has been developed thanks to funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 657956.
Proper citation: John Palmer 2018. Human Trajectory Sampling at the City-Day Scale. protocols.io dx.doi.org/10.17504/protocols.io.nj5dcq6 Copy
Authors: Bianca Hemmeryckx, Dries Bauters, H. Roger Lijnen
Summary: ADAMTS5, the main aggrecanase, is a protein that seems to play a functional role in the development of brown and white adipose tissue (WAT) and browning of WAT. These published observations were made using the ADAMTS5-P mice originally generated by Pfizer, in collaboration with Lexicon Genetics (The Woodlands, TX, USA).
Proper citation: Bianca Hemmeryckx, Dries Bauters, H. Roger Lijnen 2018. Genotyping the ADAMTS5-P (Pfizer) mouse strain. protocols.io dx.doi.org/10.17504/protocols.io.kaucsew Copy
Authors: Takao Ito, Ryoji Nakaune
Summary: In the STEPS, we describe TaqMan multiplex real-time PCR to universally detect phytoplasmas (PP) and Xylella spp. (XL) with plant internal control (IC) from crude extracts. A protocol file uploaded in the DESCRIPTION shows further details of the protocol in Japanese and English.
Proper citation: Takao Ito, Ryoji Nakaune 2017. A protocol of molecular detection of phytoplasmas and Xylella spp. in post-entry quarantine for plants.. protocols.io dx.doi.org/10.17504/protocols.io.jhjcj4n Copy
Authors: Alle Lie
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology
Proper citation: Alle Lie 2016. The culturing of axenic Ochromonas sp. strain CCMP1393. protocols.io dx.doi.org/10.17504/protocols.io.ey8bfzw Copy
Authors: Jenny T. van der Steen, Mirjam C. van Soest-Poortvliet, Cornelis A. van den Bogert, René H.J. Otten, Gerben ter Riet, Lex M. Bouter
Summary: Background: Selective reporting distorts the aggregate body of scientific evidence, wastes resources and can harm patients’ health and the credibility of science. Selective reporting may result from a focus on preferred findings by researchers and others stakeholders. However, it is unclear if some persons or environments are at greater risk of selective reporting than others. Our review assesses what is known and what is postulated about determinants of selective reporting in the scientific domain.Methods/design: Using search terms for bias and selection combined with terms for reporting and publication, we systematically search the PubMed, Embase, PsycINFO and Web of Science databases. A 25 percent random selection of records is reviewed for inclusion by at least two reviewers based on title and abstract if available and, if needed, the full text. Inter-rater agreement is calculated. Examining the content of the entire article, including, for example the discussion section, we extract phrases mentioning determinants. From this, we compile a structured list of possible determinants. The results are also categorized by type of source (empirical result or view, study design, and academic discipline if variability suffices). We follow the PRISMA-P and PRISMA reporting guidelines for systematic reviews where applicable.Discussion: We use principles of the systematic review along with principles of qualitative content analysis to focus on the nature of possible determinants of selective reporting. Our review includes both empirical findings and theoretical considerations. It will identify priority questions for further research on mechanisms of selective reporting. Furthermore, it will help in assessing risk of selective reporting. Such “risk profiling” may inform effective policy development on responsible conduct of research. The approach of combining quantitative and qualitative assessment techniques is expected to optimally inform well-targeted intervention studies and inspire policy development.
Proper citation: Jenny T. van der Steen, Mirjam C. van Soest-Poortvliet, Cornelis A. van den Bogert, René H.J. Otten, Gerben ter Riet, Lex M. Bouter 2017. Determinants of selective reporting: protocol for a review and content analysis of a random selection of the literature . protocols.io dx.doi.org/10.17504/protocols.io.jz3cp8n Copy
Authors: Alise Ponsero
Group: Metafunc course 2018
Summary: This protocol explores how to download datasets from the HMP portal, either from a sample id number.
Proper citation: Alise Ponsero 2018. Download datasets from the HMP portal. protocols.io dx.doi.org/10.17504/protocols.io.srzed76 Copy
Authors: Deborah A. Howatt, Anju Balakrishnan, Hong Lu
Group: Sangderk Lee lab
Proper citation: Deborah A. Howatt, Anju Balakrishnan, Hong Lu 2017. Blood Pressure Measurement: Kent Coda 8 (Tail-Cuff). protocols.io dx.doi.org/10.17504/protocols.io.ix6cfre Copy
Authors: Alba Balletbó
Group: iGEM Wageningen 2019
Proper citation: Alba Balletbó 2019. Electrocompetent E. coli . protocols.io dx.doi.org/10.17504/protocols.io.7jrhkm6 Copy
Authors: Sam Li
Group: BioLegend
Summary: Product description and procedure summary: The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.
Note: This procedure is optimized for the isolation of 107 to 2 x 108 cells per tube. If working with fewer than 107 cells, keep volumes as indicated for 107 cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.Scale up volumes if using 14mL tubes and Magnet, and place the tube in the magnet for 10 minutes.
Sample Preparation: Enzymatic digestion of mouse spleen is recommended to achieve the highest purity and yield of CD11c+ cells. There are several protocols published that can be applied. As a general guideline, cut mouse spleen into pieces and incubate in 0.5 mg/ml Collagenase for 30 to 60 minutes at room temperature or 37°C. Place the tube in a rocking platform with continuous agitation or gently pipette every 10 minutes. Alternatively, inject 1 ml of enzymes solution in the uncut organ. Force the tissue through a 70µm filter to prepare a single cell suspension, and wash with complete media. Resuspend cells in 0.1 mg/ml DNase 1 solution and incubate at room temperature for 10 minutes. Again, filter cells through a 70 µm filter and wash with complete media. Resuspend in complete media or MojoSort™ Buffer and keep on ice until ready to use.
Proper citation: Sam Li 2019. MojoSort™ Mouse CD11c Nanobeads Protocol. protocols.io dx.doi.org/10.17504/protocols.io.7xjhpkn Copy
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