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Authors: Lisa Mesrop, Sarah Hu
Group: Caron Lab - Protistan Ecology
Summary: Protocol for PCR amplification of extracted cDNA or DNA and downstream library preparation for MiSeq sequencing.See other related protocols for extraction of RNA and/or DNA from environmental samplesThis protocol is specific to 18S rRNA gene tag sequencing targeting to V4 hypervariable region. We use V4 primers with Illumina adapters that then anneal to the P5 & P7 Illumina-specific indices. This barcoding step allows us to multiplex many samples for MiSeq sequencing. The V4 region is ~400bp, so we typically do MiSeq 250x250bp PE sequencing. 

Proper citation: Lisa Mesrop, Sarah Hu 2019. 18S V4 tag sequencing PCR amplification and library prep (Illumina). protocols.io https://dx.doi.org/10.17504/protocols.io.rfvd3n6 Copy   

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Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: Protocol for isolation of nuclei from microdissected mouse brain tissue sections for FPCR and/or RNA-seq analysis. 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 2021. Isolation of Nuclei from Adult Mouse Brain Tissue Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.bq7emzje Copy   

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Authors: New England Biolabs
Group: New England Biolabs (NEB)
Summary: The NEBNext Ultra II Directional RNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of RNA into high quality directional (strand-specific) libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow has minimal hands-on time and is compatible with poly(A) mRNA enrichment and rRNA depletion methods.

Proper citation: New England Biolabs 2018. Protocol for use with FFPE RNA, NEBNext rRNA Depletion Kit (Human/Mouse/Rat) (NEB #E6310) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760, E7765). protocols.io https://dx.doi.org/10.17504/protocols.io.tumenu6 Copy   

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Authors: Ruth Timme, Maria Balkey, William Wolfgang, Errol Strain
Group: GenomeTrakr
Summary: PURPOSE: Guidance on how to populate NCBI's Pathogen metadata package, maximizing interoporability for foodborne pathogen surveillance. SCOPE: This protocol provides detailed instructions for populating the core metadata fields within the Pathogen metadata package.

Proper citation: Ruth Timme, Maria Balkey, William Wolfgang, Errol Strain 2020. Populating the NCBI pathogen metadata template. protocols.io https://dx.doi.org/10.17504/protocols.io.be9gjh3w Copy   

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Authors: Jianlong Li

Proper citation: Jianlong Li 2017. Quantitative assessments of water-use efficiency in Temperate Eurasian Steppe along an aridity gradient. protocols.io https://dx.doi.org/10.17504/protocols.io.h3mb8k6 Copy   

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Authors: Magdalena Julkowska, Like Fokkens
Group: Salt Lab KAUST
Summary: This in silico protocol allows to have an insight into the sequence divergence of selected genomic region, including missing information, gaps and allignment with Open Reading Frames. The protocol is written for model plant Arabidopsis thaliana, but can be possibly adapted to plotting the divergence of any organism, for which multiple accessions were resequenced. We used this script to produce divergence plots in our paperMagdalena M. Julkowska, Karlijn Klei, Like Fokkens, Michel A. Haring, M. Eric Schranz, Christa Testerink; Natural variation in rosette size under salt stress conditions corresponds to developmental differences between Arabidopsis accessions and allelic variation in theLRR-KISSgene,Journal of Experimental Botany, Volume 67, Issue 8, 1 April 2016, Pages 2127–2138,https://doi.org/10.1093/jxb/erw015

Proper citation: Magdalena Julkowska, Like Fokkens 2018. PlotDivergence for plotting the natural variation in sequence identity of the chosen genetic locus encompassing multiple ORFs. protocols.io https://dx.doi.org/10.17504/protocols.io.t2ieqce 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. Monocytes, epithelium, and muscles make IL-15. It acts on T cells and activated B cells. It causes the proliferation of both B and T cells. It causes NK cell memory and CD8+ T cell proliferation. [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-15 in human serum.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj3ekqje Copy   

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Authors: Nancy Smith, Aliya Spigelman, Haopeng Lin, Patrick Macdonald
Group: CIRTNR2FIC
Summary: This protocol details islet isolation from mouse pancreas. The protocol is divided into 3 main parts; in situ perfusion of pancreas with collagenase, pancreas digestion, and islet purification. A link is provided for the purification using histopque gradient.

Proper citation: Nancy Smith, Aliya Spigelman, Haopeng Lin, Patrick Macdonald 2020. Mouse Pancreatic Islet Isolation. protocols.io https://dx.doi.org/10.17504/protocols.io.sqaedse Copy   

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Authors: Xin Liu
Group: GigaScience Press, BGI
Summary: This protocol is used to clarity the process of total DNA extration for our Betta splendens genome.

Proper citation: Xin Liu 2018. DNA extration for the Betta splendens genome. protocols.io https://dx.doi.org/10.17504/protocols.io.qq6dvze Copy   

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Authors: Jiaxin Li

Proper citation: Jiaxin Li 2020. PCR. protocols.io https://dx.doi.org/10.17504/protocols.io.bniemcbe Copy   

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Authors: Kenneth M. Stedman, Kate Porter, and Mike L. Dyall-Smith
Group: VERVE Net
Summary: These are protocols from: Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7Please see the published manuscript for additional information.

Proper citation: Kenneth M. Stedman, Kate Porter, and Mike L. Dyall-Smith 2016. The Isolation of Viruses Infecting Archaea. protocols.io https://dx.doi.org/10.17504/protocols.io.ebibake Copy   

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Authors: Dominik Metz, Sara Chiurchiù, Emanuele Bottosso, Giuseppe Pontrelli, Martina Mazzocco, Lucilla Ravà
Summary: Project Summary Background Rationale Neonatal hypoglycaemia is a common condition, particularly affecting LBW newborns, and is associated with both acute and long standing complications. Ideally, the mother can provide breast milk as soon as possible; however, in many circumstances (poor nutrition of mother, post-operative Caesarian section, etc) this may not be the case for hours or even days. If resources are available, this can be managed by providing the LBW neonate with artificial milk/donor whilst waiting for breast milk when required. However, this is often not available in resource poor countries due to expense and difficulty in production (timely clean safe water, training in how to make the milk etc). Furthermore, in resourced hospitals, glucose is monitored in at risk babies, and should they develop hypoglycemia, IV 10% dextrose can be relatively simply administered. In resource poor settings, this is challenging- firstly, glucose monitoring and then the administration of IV 10% dextrose require resources, skills and nursing that are often not available. The primary aim of this study is to assess whether the use of oral 10% dextrose, is tolerable and reduces the incidence of hypoglycaemia in LBW newborns in the absence of sufficient maternal breast milk. 10% dextrose is readily available, inexpensive and can be stored at room temperature. Objectives Our aim was to assess whether hypoglycaemia could be prevented in LBW babies with insufficient maternal breast milk (MBM), by supplementing their feed with oral 10% dextrose. Methods This is a 8 months, interventional, prospective, single centre, randomised, double arm control trial on 74 newborns conducted at a district hospital in Tanzania (Tosamaganga Hospital) where Doctors with Africa - CUAMM has ongoing projects on maternal-neonatal health. LBW newborns fulfilling the inclusion criteria will be randomly allocated to either the intervention (supplemental oral 10% dextrose) or monitoring arm. Glucose will be monitored regularly in both arms and the incidence of hypoglycaemic episodes recorded together with normal vital signs. A budget of 16,000,000Tsh has been proposed for the study. Outcomes The primary outcome will be hypoglycemia (

Proper citation: Dominik Metz, Sara Chiurchiù, Emanuele Bottosso, Giuseppe Pontrelli, Martina Mazzocco, Lucilla Ravà 2018.  Prevention of hypoglycaemia in a resource-poor setting with oral 10% dextrose in low birth weight newborns: A randomized controlled trial . protocols.io https://dx.doi.org/10.17504/protocols.io.qijducn Copy   

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Authors: Andrew Baker and Ross Cunning
Summary: Sodium acetate solution is used in DNA extraction. This recipe makes 50 mL of a 3 M stock solution at ph 5.2  -- dilute tenfold in water to a working concentration of 0.3 M for use in the DNA extraction protocol.

Proper citation: Andrew Baker and Ross Cunning 2016. Sodium acetate solution. protocols.io https://dx.doi.org/10.17504/protocols.io.dyu7wv Copy   

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Authors: Oxford HCA, Nicholas Provine, Michael FitzPatrick, Sophie Irwin
Summary: This protocol is for the isolation of the epithelial cells (and associated immune cells within the epithelial layer) from frozen human intestinal pinch biopsies, for use in single-cell sequencing or flow cytometry experiments. This protocol is adapted from Parikh, Antanaviciute, Fawkner-Corbett et al., (2019) Natuer (doi: 10.1038/s41586-019-0992-y). It has been modified specifically with the aim of increasing cell recovery from biopsies of the small intestine.

Proper citation: Oxford HCA, Nicholas Provine, Michael FitzPatrick, Sophie Irwin 2020. Isolation of cells from the epithelial layer of frozen human intestinal biopsies. protocols.io https://dx.doi.org/10.17504/protocols.io.bcb6isre Copy   

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Authors: Angel Justiz-Vaillant
Group: University of the West Indies, [email protected]
Summary: RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) also known as CCL5 is a chemotactic cytokine for T cells, basophils, and eosinophils. RANTES also acts as a recruitment signal for immune cells into inflammatory sites.

Proper citation: Angel Justiz-Vaillant 2020. ELISA for quantification of RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) in human serum or plasma.. protocols.io https://dx.doi.org/10.17504/protocols.io.bj66krhe Copy   

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Authors: James Kirkbride, Samantha Harris, Jennifer Dykxhoorn, Christina Dalman, Anna-Clara Hollander
Summary: BackgroundRefugees have reported living in extreme conditions of poverty, and the negative impact this has on their psychological well-being (Freedom from Torture report, 2013). Previous literature suggests that refugees are at a higher risk of developing psychosis as opposed to natives as well as non-refugee migrants (Hollander et al., 2016). We do not yet have a clear idea of their risk of substance abuse, although it has been shown that this is a risk people face who have suffered traumatic experiences (Stewart, 1996). The objective of this study is to determine whether refugees and non-refugee migrants are at an elevated risk of substance misuse relative to the Swedish-born population. We will consider the impact of PTSD on the association between refugee status and substance use disorders.MethodSettingSweden is said to have taken in more refugees than any other European country, making it an ideal country to conduct this research on. The study will follow similar methods as Hollander and colleagues, who conducted their study using the Psychiatry Sweden database, a large database of linked national registers that provides a variety of information on everyone living in Sweden, such as date of birth, sex and country of birth. MeasuresUsing the information from the database, it will be possible to calculate hazard ratios for substance use disorders admissions in refugees, non-refugee migrants, and Swedish natives. We will also consider alcohol use, cannabis use and polydrug use disorders separately.SampleParticipants will include Swedish natives, born to two Swedish parents, non-refugee migrants, and refugees living in Sweden. The cohort will comprise of all people born between 1984-1997, living in Sweden on or after their 14thbirthday and followed until 31 December 2016, substance use disorder, emigration or death, whichever was sooner. We will restrict the cohort to refugees and non-refugee migrants from regions-of-origin where at least 1000 refugees are recorded in the Swedish total population register to permit meaningful comparisons. Study proceduresThe study will establish a prospective cohort from the database. The main exposure of interest examined will be refugee status, while the main outcome is substance misuse related admissions. People with ICD-10 substance use disorders (F10-19) will be identified from the National Patient Register. PTSD will be recorded in the NPR according to ICD-10 43.1. Possible confounders will be considered, such as: age at arrival, age and sex, disposable income employment status and population density, as these may all be related to substance misuse. Approach to analysisWe will gather basic descriptive statistics, and crude incidence rates for each of the three groups. Similarly to Hollander and colleagues, we will fit Cox proportional hazards models in order to calculate hazard ratios and confidence intervals. PTSD will be considered as a confounder and effect modifier of the refugee-substance use disorder association. References:o Hollander, A.-C., Dal, H., Lewis, G., Magnusson, C., Kirkbride, J. B., & Dalman, C. (2016). Refugee migration and risk of schizophrenia and other non-affective psychoses: cohort study of 1.3 million people in Sweden. Bmj-British Medical Journal, 352. doi:10.1136/bmj.i1030o Stewart, S. H. (1996). Alcohol abuse in individuals exposed to trauma: A critical review. Psychological Bulletin, 120(1), 83-112. doi:10.1037//0033-2909.120.1.83o Freedom from Torture report, Jo Pettitt, 2013

Proper citation: James Kirkbride, Samantha Harris, Jennifer Dykxhoorn, Christina Dalman, Anna-Clara Hollander 2019. Admission for substance-induced disorders in refugee and migrant groups in Sweden. protocols.io https://dx.doi.org/10.17504/protocols.io.53fg8jn Copy   

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Authors: Karen Maciel de Oliveira
Summary: Mitochondrial viability is determined through 2,3,5-triphenyltetrazolium chloride (TTC) conversion to insoluble formazan. TTC reduction depends on the mitochondrial respiratory activity and is proportional to the number of viable cells.

Proper citation: Karen Maciel de Oliveira 2018. Mitochondrial viability. protocols.io https://dx.doi.org/10.17504/protocols.io.ruud6ww Copy   

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Authors: Binnypreet Kaur1, 2, Drahomíra Faktorová1, 2 and Julius Lukeš1, 2, 3
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Julius Lukes
Summary: The process of NHEJ is completed when the DNA ligase complex ligates the broken ends of the DNA. Ku70-Ku80 are the DNA-dependent protein kinase heterodimeric regulatory factor that forms a link between two broken DNA ends and structurally support, align and protect them from further degradation. W7 inhibitor (W7(N-(6-aminohexyl)-5-chloro1-naphthalenesulfonamide) inhibits the cofactor of inositol hexakisphophate which effects the regulatory factor of Ku heterodimers.

Proper citation: Binnypreet Kaur1, 2, Drahomíra Faktorová1, 2 and Julius Lukeš1, 2, 3 2019. Transfectionof constructs in diplonemids to block NHEJ pathway using the inhibitors of KU70/80 proteins.. protocols.io https://dx.doi.org/10.17504/protocols.io.x35fqq6 Copy   

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Authors: Martha Campbell Thompson, Malavika Nair, Aidan Sullivan
Group: SPARC

Proper citation: Martha Campbell Thompson, Malavika Nair, Aidan Sullivan 2020. Vesselucida 360 Protocol for Segmenting and Analyzing Human Islet Microvasculature. protocols.io https://dx.doi.org/10.17504/protocols.io.bjfzkjp6 Copy   

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Authors: Robert Hooftman
Group: iGEM Wageningen 2019

Proper citation: Robert Hooftman 2019. Chitin binding assay using chitin magnetic beads (NEB). protocols.io https://dx.doi.org/10.17504/protocols.io.8anhsde Copy   

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