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Authors: Elena Hilario, Marcus Davy, Roy Storey, Richard Newcomb, Ross N Crowhurst, Roger P Hellens
Summary: A genome is assembled by connecting large scaffolds via a collection of thousands of mate-paired ends of various sizes (3-20 kbp). Terminal ends of long DNA fragments can be obtained by sequencing large insert clones by Sanger sequencing, a labour-intensive and expensive approach. Here we describe a high-throughput protocol to tag 11,520 BAC plasmids randomly with barcoded oligonucleotides at the end of a random hexamer by PCR.This method assisted the kiwifruit (Actinidia chinensis) genome assembly in a similar way as mate paired end datasets.  Although not fully sequenced, each randomly tagged BAC clone provides a collection of short sequence tags mapped to a genome across one or multiple scaffolds under three different scenarios: validate a single scaffold, join two scaffolds, or group several scaffolds by proximity only. Our method provides a simple way of cataloging BAC libraries to assist genome assembly projects.

Proper citation: Elena Hilario, Marcus Davy, Roy Storey, Richard Newcomb, Ross N Crowhurst, Roger P Hellens 2018. BACRB - Parallel Sequencing of Barcoded BAC Clones by random tagging PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.p3adqie Copy   

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Authors: HANNIGAN GD, GRICE EA, ET AL.
Group: VERVE Net, Club Grice
Summary: This protocol outlines the analysis used to plot contig coverage statistics, as well as sequence count and length stats. We begin with visualizing contig length vs coverage. We then visualize the distributions of sequence counts per sample as a probability density plot (similar idea as a histogram), and then do the same for median sequence length. Based on the methods from the following publication:Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." mBio 6.5 (2015): e01578-15.

Proper citation: HANNIGAN GD, GRICE EA, ET AL. 2016. Script R1: Virome Contig and Sequencing Statistics. protocols.io https://dx.doi.org/10.17504/protocols.io.eh5bb86 Copy   

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Authors: Chisholm Lab
Group: VERVE Net, Sullivan Lab

Proper citation: Chisholm Lab 2016. NATURAL SEAWATER-BASED PRO99 MEDIUM. protocols.io https://dx.doi.org/10.17504/protocols.io.c7zzp5 Copy   

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Authors: Grossman Lab, Pringle Lab
Group: Aiptasia-Symbiodiniaceae Model System
Summary: This protocol describes briefly how to perform colonization of aposymbiotic Aiptasia with Symbiodinium cultures.The method is based on the one described in Xiang et al., 2013.

Proper citation: Grossman Lab, Pringle Lab 2018. Colonization of aposymbiotic Aiptasia with Symbiodinium. protocols.io https://dx.doi.org/10.17504/protocols.io.rh4d38w Copy   

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Authors: Lukas Snoek, Tinka Beemsterboer
Group: Spinoza Centre, REC-L
Summary: This protocol lists all the steps necessary to run your MRI experiment/data acquisition safely and in a way that yields high-quality data. Moreover, if you use the centre's QC/preprocessing service, it lists the steps necessary to make sure we can convert the data into BIDS and run them through the QC/preprocessing pipelines.

Proper citation: Lukas Snoek, Tinka Beemsterboer 2018. During data acquisition. protocols.io https://dx.doi.org/10.17504/protocols.io.p5vdq66 Copy   

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Authors: Christoph Bott, Ingo Dressel, Peter Bayer
Summary: This supplement to the article "Paraffin wax as self-sealing insulation material for seasonal sensible heat storage systems – A laboratory study" contains the temperature data recorded in the laboratory for section 2.1 "Enhancement of thermal performance".

Proper citation: Christoph Bott, Ingo Dressel, Peter Bayer 2020. Paraffin wax as self-sealing insulation material for seasonal sensible heat storages - Enhancement of thermal performance - Temperature Records. protocols.io https://dx.doi.org/10.17504/protocols.io.bg8ejzte Copy   

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Authors: Vinay Saunders, David I Wilson
Summary: Protocol describing fluorescence immunohistochemistry for detection of thymosin beta 4 alongside CD31, smooth muscle actin and myosin heavy chain in PFA-fixed human cardiac tissue samples.

Proper citation: Vinay Saunders, David I Wilson 2018. TB4 Fluorescence Immunohistochemistry. protocols.io https://dx.doi.org/10.17504/protocols.io.rwcd7aw Copy   

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Authors: Jeffrey Lewis, Amanda Scholes
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Lewis Lab
Summary: A generalizable protocol for measuring relative changes in gene expression via qPCR (cDNA synthesis, qPCR primer optimization, and qPCR analysis), with specific optimization for the budding yeast Saccharomyces cerevisiae.

Proper citation: Jeffrey Lewis, Amanda Scholes 2020. Quantitative (q)PCR and Differential Expression Analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.bbgpijvn Copy   

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Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol is used for AAV production in HEK293T cells. To see the full abstract and additional resources, visit the Addgene protocol page.Sample Data

Proper citation: Addgene The Nonprofit Plasmid Repository 2019. AAV Production in HEK293T Cells. protocols.io https://dx.doi.org/10.17504/protocols.io.bawpifdn Copy   

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Authors: Peter Havel
Group: Mouse Metabolic Phenotyping Centers
Summary: Summary: Albumin blue dye is a stain for the specific and sensitive spectrofluorometric determination of albumin in natural matrices. AB 580 binds to the albumin present in urine samples and the fluorescence can be quantified using a fluorimeter.

Proper citation: Peter Havel 2019. UC Davis - Uninary Albumin Excretion (UAE) Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.yw5fxg6 Copy   

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Authors: James Skelton, Craig Bateman, Jiri Hulcr
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol describes how to extract ambrosia fungi from Xylosandrus compactus, the black twig borer. This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 https://doi.org/10.1007/s13199-020-00686-9.

Proper citation: James Skelton, Craig Bateman, Jiri Hulcr 2020. Fungal extraction from Xylosandrus compactus. protocols.io https://dx.doi.org/10.17504/protocols.io.bnuumeww Copy   

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Authors: Jacob Harris
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Alverson Lab
Summary: The FlowCam can be used to take quick cell density measurements of liquid samples. 

Proper citation: Jacob Harris 2016. FlowCam Standard Operating Procedure. protocols.io https://dx.doi.org/10.17504/protocols.io.f2tbqen Copy   

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Authors: Longzhi Tan

Proper citation: Longzhi Tan 2020. Isolation and Fixation of Nuclei from the Mouse Brain for Dip-C. protocols.io https://dx.doi.org/10.17504/protocols.io.bpsxmnfn Copy   

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Authors: LI-COR Biosciences
Group: LI-COR Biosciences
Summary: Developed for: Aerius, Odyssey® Classic, Odyssey CLx, and Odyssey Sa Infrared Imaging Systems

Proper citation: LI-COR Biosciences 2018. Complete Sample Protocol for Measuring IC50 of Inhibitor PD168393 in A431 Cells Responding to Epidermal Growth Factor. protocols.io https://dx.doi.org/10.17504/protocols.io.grxbv7n Copy   

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Authors: Shuangshuang Li

Proper citation: Shuangshuang Li 2018. Drug sensitivity assay (CCK8). protocols.io https://dx.doi.org/10.17504/protocols.io.h4mb8u6 Copy   

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Authors: Lian He, Yue-He Ding, Peng Tan, Ji Jing, Meng-Qiu Dong, Yubin Zhou
Summary: Specialized junctional sites that connect the plasma membrane(PM) and endoplasmic reticulum(ER) are intimately involved in controlling lipid metabolism and calcium signaling in mammalian cells. Store operated calcium entry mediated by dynamic STIM1-ORAI1 coupling constitutes one of the most well-established molecular events occurring at ER-PM junctions, but the protein composition at this particular subcellular compartment remain poorly defined. Using an in situ spatially-restricted biotin-labeling coupled with mass spectrometry, we mapped the proteome of intact ER-PM junctions in living cells without disrupting their architectural integrity. Our approaches lead to the discovery of >70 candidate proteins at ER-PM junctions, with the majority falling into the categories of ER/PM-resident proteins, cytoskeletal components, and proteins functioning in intracellular membrane trafficking or post-translational modifications. Although the current protocol is limited to ER-PM junctions, the methods described herein can be readily extended to study other types of inter-membrane appositions in various types of cells.

Proper citation: Lian He, Yue-He Ding, Peng Tan, Ji Jing, Meng-Qiu Dong, Yubin Zhou 2019. Proteomic mapping of ER-PM junctions in living HEK293 cells. protocols.io https://dx.doi.org/10.17504/protocols.io.3kxgkxn Copy   

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Authors: Udani Wijewardhana, Denny Meyer, Madawa Weerasinghe
Summary: AbstractBackgroundDue to certain risk factors such as climate change, habitat destruction, overhunting and pollution bird extinctions are now occurring at a rate that far exceeds their speciation rate. When a bird species is lost, the benefits it might have afforded are gone forever. There are no robust indicators of biodiversity conservation that can be used to complement existing national indicators of economic and social health. This study reviews the statistical methods that are used to model and evaluate the persistence or extinction risk of threatened bird species using citizen science data.MethodsAdhering to PRISMA guidelines, this review systematically searched for relevant journal articles which were published between January 1900 and January 2019 in any of three databases (ProQuest Central, Scopus and Web of Science). Only the journal articles which used a statistical model, predictive model or trend analysis in analysing persistence or extinction risk (while considering risk factors) of threatened bird species developed using citizen science data were included in this study. The bird species that may be declining in population/range even in near threatened or least concern categories also included since these may be the next wave of species to be added to the endangered species lists. Citizen science data helps to increase the number of records and understanding of dynamics in declining bird species populations.ResultsThis systematic review was able to identify 37 unique articles describing statistical models for this purpose. Generalised linear models and linear mixed/hierarchical models were the most popular methods used for analysis, followed by machine learning models. The review suggested several strategies to measure the persistence of threatened bird species, but there was no attempt to identify critical tipping points using methods such as change-point analysis.ConclusionOverall, it appears that the persistence of threatened bird species varies depending on various risk factors. Statistical models can provide a better understanding of the impact of these risk factors, which is expected to produce better immediate and long-term outcomes for the conservation of threatened birds. It is hoped that this review will identify the statistical methods which are most suitable for this purpose.

Proper citation: Udani Wijewardhana, Denny Meyer, Madawa Weerasinghe 2019. Statistical methods to analyze the persistence of threatened birds using citizen science data: A systematic review. protocols.io https://dx.doi.org/10.17504/protocols.io.3dagi2e Copy   

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Authors: Ken Stedman, Geoffrey Diemer, Jennifer E Kyle
Group: VERVE Net
Summary: An alternative epifluorescence microscopy (EFM) technique for virus enumeration has been developed using polycarbonate Track Etch (PCTE) filter membranes in place of aluminum oxide Anodisc membranes. The established EFM technique, first developed by Hennes and Suttle in 1995 and later expanded upon by Noble and Fuhrman in 1998, uses supported 20 nm pore-size Anodisc filter membranes to determine virus abundance in natural environments. Increased price and sporadic availability of Anodisc filters stimulated the evaluation of alternative filters for use in the procedure. The feasibility of using 30 nm pore-size PCTE filters for virus enumeration was assessed using the Anodisc filter procedure as a control. Although virion particle counts are slightly less precise using PCTE filters, they offer a substitute for Anodiscs while requiring only minor adjustments to the established protocol. Per slide, the PCTE costs approximately ten times less to prepare than the Anodisc-based method.

Proper citation: Ken Stedman, Geoffrey Diemer, Jennifer E Kyle 2016. PCTE Protocol for VLP enumeration. protocols.io https://dx.doi.org/10.17504/protocols.io.eaubaew Copy   

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Authors: Hongbo Zhang
Summary: This protocol describes a methond of single nucleus isolaton from human skeletal muscle for snRNA sequencing.

Proper citation: Hongbo Zhang 2018. Nuclei isolation from human skeletal muscle. protocols.io https://dx.doi.org/10.17504/protocols.io.t68erhw Copy   

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Authors: Jennifer Rutkowsky
Group: Mouse Metabolic Phenotyping Centers
Summary: Ionized calcium-binding adapter molecule 1 (IBA1) is specifically expressed in macrophages / microglia and is upregulated during the activation of these cells. Iba1 expression is up-regulated in microglia following nerve injury,[4] central nervous system ischemia, and several other brain diseases. Furthermore it has been found in athlesclerotic plaques and at sigts of vascular injury. Modified from: IHC Methods and Materials VMTH - Anatomic Pathology, UC-Davis

Proper citation: Jennifer Rutkowsky 2019. UC Davis - Immunohistochemistry IBA1. protocols.io https://dx.doi.org/10.17504/protocols.io.56pg9dn Copy   

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