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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. Il-38 acts on T cells and inhibits the synthesis of IL-17 and IL-22. The placenta, tonsil's B lymphocytes, spleen, skin, and thymus widely express IL-38. [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-38 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj4dkqs6 Copy   

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Authors: Sonia M. Hall

Proper citation: Sonia M. Hall 2016. Optimized Concentrations for Developmental Studies Hybridoma Bank and Custom Antibodies for use with Drosophila Embryos - HeLa Cell Additions. protocols.io https://dx.doi.org/10.17504/protocols.io.fewbjfe Copy   

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Authors: Ian Mackay
Summary: This is an excellent rhinovirus screening assay which I have used on thousands of sample extracts mostly originating from acutely ill paediatric patients, spanning well over a decade's worth of collection dates.I have not confirmed that it can detect every single genotype but I do know that it detects many from each of the three RV species (Human rhinovirus A, Human rhinovirus B and Human rhinovirus C) as well as at least some Human enterovirus genotypes.The assay does pick up some enteroviruses due to their genetic similarities in the 5'UTR target region. These can be discriminated using subgenomic sequencing (see  VP42 typing assay protocol), or simply described as "respiratory enteroviruses" since there is no specific-specific vaccine or treatment available anyway. The assay was created and first described in 2008 by Xiaoyan Lu and colleagues at the US CDC [Ref 1]. In collaboration with Lu et al., we published a small update that included Lu's modified forward primer design. We routinely use it with this primer (hence the 'mod' text added to the primer name) but without the expensive special base, as outlined in Step 1.  

Proper citation: Ian Mackay 2018. Human rhinovirus screening real-time RT-PCR ("modified Lu assay"). protocols.io https://dx.doi.org/10.17504/protocols.io.nz4df8w Copy   

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Authors: Karen D. Weynberg, Elisha M. Wood-Charlson, Curtis A. Suttle, and Madeleine J. H. van Oppen
Group: VERVE Net
Summary: Reef-building corals comprise multipartite symbioses where the cnidarian animal is host to an array of eukaryotic and prokaryotic organisms, and the viruses that infect them. These viruses are critical elements of the coral holobiont, serving not only as agents of mortality, but also as potential vectors for lateral gene flow, and as elements encoding a variety of auxiliary metabolic functions. Consequently, understanding the functioning and health of the coral holobiont requires detailed knowledge of the associated viral assemblage and its function. Currently, the most tractable way of uncovering viral diversity and function is through metagenomic approaches, which is inherently difficult in corals because of the complex holobiont community, an extracellular mucus layer that all corals secrete, and the variety of sizes and structures of nucleic acids found in viruses. Here we present the first protocol for isolating, purifying and amplifying viral nucleic acids from corals based on mechanical disruption of cells. This method produces at least 50% higher yields of viral nucleic acids, has very low levels of cellular sequence contamination and captures wider viral diversity than previously used chemical-based extraction methods. We demonstrate that our mechanical-based method profiles a greater diversity of DNA and RNA genomes, including virus groups such as Retro-transcribing and ssRNA viruses, which are absent from metagenomes generated via chemical-based methods. In addition, we briefly present (and make publically available) the first paired DNA and RNA viral metagenomes from the coral Acropora tenuis.

Proper citation: Karen D. Weynberg, Elisha M. Wood-Charlson, Curtis A. Suttle, and Madeleine J. H. van Oppen 2016. Generating viral metagenomes from the coral holobiont. protocols.io https://dx.doi.org/10.17504/protocols.io.ejgbcjw Copy   

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Authors: Elliott Swanson, Lucas Graybuck, Pete Skene
Group: Human Cell Atlas Method Development Community
Summary: TEA-seq is a method for Transcriptomic, Epitope, and Accessibility measurement from thousands of single cells on the 10x Genomics Multiome platform. It generates scRNA-seq, scATAC-seq, and cell surface epitope (BioLegend TotalSeq) sequencing libraries linked by 10x cell barcodes for coordinated analysis. TEA-seq has been optimized for use with peripheral blood mononuclear cells (PBMCs).

Proper citation: Elliott Swanson, Lucas Graybuck, Pete Skene 2020. TEA-seq. protocols.io https://dx.doi.org/10.17504/protocols.io.bqagmsbw Copy   

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Authors: Tyler C. Moore
Group: Bellevue University Natural Sciences Laboratories
Summary: Counting cells with a hemocytometer is an easy way to determine relatively accurate numbers of viable cells.  After determining cell counts, cells can be passaged, frozen away, or used for an experiment at a particular density.  

Proper citation: Tyler C. Moore 2018. Counting Cells with Hemocytometer. protocols.io https://dx.doi.org/10.17504/protocols.io.nxsdfne Copy   

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Authors: Jiri Hulcr, You Li
Group: Protocols Bark Beetle Mycobiome
Summary: This protocol describes how to estimate the number of colony-forming units.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: Jiri Hulcr, You Li 2020. Calculating Colony-forming Units. protocols.io https://dx.doi.org/10.17504/protocols.io.bnuvmew6 Copy   

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Authors: John Tyson
Summary: Bead-Free LSK109 ligation prep for ultra-long DNAThe use of AMPureXP beads for reaction cleanup when targeting Ultra-long DNA reads is not optimal as inter-bead binding results in bead clumping and potential fragmentation as individual beads move around freely in suspension. This is why the original Ultra-read method developed by Josh Quick and Nick Loman used dilution of the Rapid MuA kit and high concentrations of HMW genomic DNA. Saturating the MuA insertion reaction with HMW DNA results in less individual strand fragmentation and produces previously unattainable read lengths. It does not require cleanup or binding and elution on and off magnetic beads and minimized “handling” of the DNA. This therefore mitigates the fragmentation and DNA recovery issue with very long DNA molecules and has allowed the production of individual reads in excess of 1 million bases to be produced. The yield from individual flowcells using this approach is around an order of magnitude less however as there appears to be flowcell surface damage and rapid pore “blockage”. While 1Mb+ reads are amazing to see, they are a very small fraction of the total and it is the read N50 that is important. Targeting 100kb+ reads in volume goes a long way to producing substantial improvements in large genome assemblies particularly when trying to span large repeats or low complexity regions refractory to other sequencing technologies. We have been experimenting with DNA size fractionation / precipitation using PEG/NaCl as mentioned above. This provides a means to remove the requirement for magnetic beads with their associated issues, and allows a higher yield of ultra-long reads to be produced. We have found that the DNA produced from the Phenol/Chloroform prep above provides DNA of a size that requires shearing down into a range to provide a mix of length and yield. Shown below is data from a single flowcell that I ran multiple libraries on looking at the effects of sequential shearing to gradually shorter lengths using a single DNA sample and where aliquots were removed before the next smaller shear was performed. Using a P1000 pipette tip or simple needle shear and PEG/NaCl precipitations, you are able to produce up to 21 % of total sequenced data in 100kb+ reads. Combined with DNaseI flowcell clears, high yields are looking obtainable. It is early days for this protocol so expect changes to come quickly as we try and dial in better size selection and an even higher proportion of 100kb+ reads. At present we are not really seeing much short read depletion and I think this is to do with suboptimal conditions used for the DNA precipitation. Unknown buffer components in the End-Prep and Ligation buffers appear to be throwing off the PEG/NaCl parameters we have identified for size selection so there is still work to be done to tune this and something we are working on. For the 21G and 29G examples below we did do a final PEG/NaCl precipitation of the finished library but didn’t see consistent depletion comparing the two, so still things to be worked out for consistency and to understanding what is happening.As a fall back, assuming little size selection can be implemented during the prep for now, we looked again at the size selection step at the end of the library preparation when eluted into EB buffer (no other salts or other components to throw size selective precipitation off). This is shown below comparing the Circulomics SRE buffer and 9 % PEG8000 (w/v) 1 M NaCl on precipitation and recovery using an equal volume addition to a P1000/26G needle sheared Bead-free LSK109 prep. Take this with a grain of salt at the moment as one used an old MinION flowcell (for Circulomics) and the others (library before selection (control), and PEG/NaCl size selection) were run on flongles so data volume is a little low with long frag blockage. We are seeing the SRE buffer performing better at short fragment removal, but want to investigate what is going on with the upper end and read N50.

Proper citation: John Tyson 2020. Bead-free long fragment LSK109 library preparation. protocols.io https://dx.doi.org/10.17504/protocols.io.7eshjee Copy   

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Authors: Robert Auber, Jennifer Wisecaver
Group: High molecular weight DNA extraction from all kingdoms, Wisecaver Lab
Summary: This is a protocol adapted for a haptophte algae. It is a single cellular organism lacking any type of silica or calcium carbonate armor. If your organism doesn't lyse by a simple snap freeze, some type of grinding step is recommended. Confirmation of properly extracting the nuclei can be done with propidium iodide staining and an epiflourescent microscope. The actual DNA extraction gives ultra long reads (greater than 200kb) by using a circulomics NanoBind kit.

Proper citation: Robert Auber, Jennifer Wisecaver 2019. Algal DNA extraction for HMW Nanopore sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.45dgy26 Copy   

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Authors: David Eccles
Summary: This protocol demonstrates how to convert raw long reads produced using a strand-specific sequencing protocol (e.g. ONT's strand-switching protocol) into strand-specific mapped reads.The general ides is to use LAST to identify the adapter orientation relative to the genome, and then use that information to reorient called sequences to create a stranded BAM files that is displayable in a genome browser.

Proper citation: David Eccles 2018. Stranded Mapping from Long Reads. protocols.io https://dx.doi.org/10.17504/protocols.io.syheft6 Copy   

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Authors: Alba Balletbó
Group: iGEM Wageningen 2019

Proper citation: Alba Balletbó 2019. SOC Medium. protocols.io https://dx.doi.org/10.17504/protocols.io.7juhknw Copy   

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Authors: Hebert F.O., Grambauer S., Barber I., Landry C.R., Aubin-Horth N.
Group: GigaScience Press
Summary: This protocol describes how the parasitic flatworms (S. solidus) were cultured in the lab for:Hebert, F, O; Grambauer, S; Barber, I; Landry, C, R; Aubin-Horth, N (2016): Reference transcriptome sequence resource for the study of the Cestode Schistocephalus solidus, a threespine stickleback parasite. GigaScience Database. http://dx.doi.org/10.5524/100197

Proper citation: Hebert F.O., Grambauer S., Barber I., Landry C.R., Aubin-Horth N. 2016. Schistocephalus solidus culturing. protocols.io https://dx.doi.org/10.17504/protocols.io.ew8bfhw Copy   

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Authors: Motoko Tanaka, Brook C Barajas, Jaisri R Lingappa
Group: Lingappa Lab Group
Summary: RNA coIP from gradients

Proper citation: Motoko Tanaka, Brook C Barajas, Jaisri R Lingappa 2017. RNA coIP from gradients. protocols.io https://dx.doi.org/10.17504/protocols.io.k74czqw Copy   

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Authors: Jeff Lewis
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Lewis Lab
Summary: A simple 96-well plate assay for stress resistance across a range of stress doses. Originally designed for yeast, but easily modifiable for any colony-forming microbe.

Proper citation: Jeff Lewis 2017. Acquired Stress Resistance Assay. protocols.io https://dx.doi.org/10.17504/protocols.io.g7sbzne Copy   

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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. Immune cells and activated epithelial cells secrete IL-20. It acts on epithelial cells. It plays a vital role in the cellular communication between epithelial cells and the immune system under inflammatory conditions.[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-20 in human serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bkewktfe Copy   

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Authors: Sam Li
Group: BioLegend

Proper citation: Sam Li 2019. Direct-Blot™ Western Blotting Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.98rh9v6 Copy   

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Authors: Judy Northill, Russell Simmons
Group: Public Health Virology, Forensic and Scientific Services
Summary: A real-time PCR method for the detection of Molluscum contagiosum virus from human samples. This assay targets the MC021L gene and detects both subtype 1 and 2 of the virus.

Proper citation: Judy Northill, Russell Simmons 2017. Molluscum contagiosum real-time PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.kkscuwe Copy   

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Authors: Mª Dolores Gelado Caballero, Tania Montoto Martínez
Summary: The following protocol is fit for seawater sample preparation prior to its observation under a stereomicroscope for the determination or microplastic particles.

Proper citation: Mª Dolores Gelado Caballero, Tania Montoto Martínez 2020. Seawater sample preparation for microplastic determination. protocols.io https://dx.doi.org/10.17504/protocols.io.bcfyitpw Copy   

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Authors: Jian Tu, Ting Xiong
Summary: Methods and results of supplement figure 1

Proper citation: Jian Tu, Ting Xiong 2019. Suppement data in AJPGI-00061-2018R2. protocols.io https://dx.doi.org/10.17504/protocols.io.x8qfrvw Copy   

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Authors: Amy Zimmerman, Camille Poirier
Group: VERVE Net, Worden Lab
Summary: Purpose: To quantify virus-like particles in culture or environmental samples.Host cells and virus-like particles are enumerated from the same glutaraldehyde-fixed sample. For virus counts, an aliquot is diluted in 0.02-µm-filtered TE buffer prior to staining with SYBR Green I nucleic acid dye to improve staining efficiency and ensure an appropriately low event rate in the instrument. An unstained aliquot of sample is run for host cell counts.

Proper citation: Amy Zimmerman, Camille Poirier 2017. Enumerating algal viruses by flow cytometry. protocols.io https://dx.doi.org/10.17504/protocols.io.guibwue Copy   

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