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Authors: Christian Boehm
Group: OpenPlant Project, Plantae
Summary: Marchantia protoplast preparation (adapted from Bopp and Vicktor 1988)
Proper citation: Christian Boehm 2016. Marchantia Protoplast Isolation. protocols.io dx.doi.org/10.17504/protocols.io.gmubu6w Copy
Authors: Vasilios Zachariadis, Huaitao Cheng, Nathanael Andrews, Martin Enge
Group: Enge lab
Summary: Understanding how genetic variation alters gene expression - how genotype affects phenotype - is a central challenge in biology. To address this question in complex cell mixtures, we developed Direct Nuclear Tagmentation and RNA-sequencing (DNTR-seq), which enables whole genome and mRNA sequencing jointly in single cells.
Proper citation: Vasilios Zachariadis, Huaitao Cheng, Nathanael Andrews, Martin Enge 2020. Direct nuclear tagmentation and RNA-sequencing (DNTR-seq). protocols.io dx.doi.org/10.17504/protocols.io.bdthi6j6 Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the instructions for setup and scanning of fluorescently-labeled tissue sections mounted in agarose and attached to a slide using the TissueCyte 1000 block face 2-photon system.
Proper citation: Allen Institute for Brain Science 2020. Core Protocol for Serial Imaging of Fluorescently-Labeled Mouse Brain on the TissueCyte 1000 System. protocols.io dx.doi.org/10.17504/protocols.io.bbx4ipqw Copy
Authors: Cecile Chardon, Valentin Vasselon, Marine Vautier, Sonia Lacroix, Agnès Bouchez, Frédéric Rimet, Isabelle Domaizon
Group: EcoALpsWater
Summary: This protocol is part of the DNA workflow applied in the Eco-ALpsWater Project, here in particular to characterize the diversity of diatom assemblage in biofilms or plankton samples.Different studies have already revealed the potential of diatom metabarcoding applications for freshwater quality assessment (Kermarrec et al. 2014; Vasselon et al. 2017ab; Visco et al. 2015). The choice of the marker gene and barcode region is key for obtaining relevant inventories of diversity and precise taxonomic assignment. For benthic diatoms, the rbcL gene has proved to be an appropriate taxonomic marker for biomonitoring (Kermarrec et al. 2013, 2014; Vasselon et al. 2017a,b) and a well‐curated barcode reference library is already available to assign species names to rbcL sequences (R‐Syst::diatom, Rimet et al. 2016).For the Eco-AlpsWater project, biolfilms sampled in rivers and lakeshores are collected as described in the dedicated protocols (“Lake plankton sample collection ...” and “Biofilms sample collection ...”) and DNA is extracted as described in the protocol “Biofilms DNA extraction” ; all these protocols are part of the Deliverable D.T1.1.2. We present here the following step in the DNA workflow (i.e. PCR amplification of selected barcodes, and wet lab methods to prepare DNA library for downstream MiSeq Sequencing). This protocol has been used in recent studies (e.g. Keck et al 2018 ; Vasselon et al 2018) where diatoms metabarcoding has been used for ecological assessment of rivers.Several primers were proposed in the literature to characterize Diatom communities through environmental DNA metabarcoding approaches, including the 18S, COI and rbcL barcodes. Following the recommendation provided by Kermarrec et al. 2014, who compared the efficiency of those 3 barcodes to accurately characterize diatom communities from freshwater samples (lakes and rivers), the rbcL barcode will be used within the Eco-AlpsWater project as he provides a good taxonomical resolution.
Proper citation: Cecile Chardon, Valentin Vasselon, Marine Vautier, Sonia Lacroix, Agnès Bouchez, Frédéric Rimet, Isabelle Domaizon 2020. Diatom DNA library preparation for Illumina Miseq Sequencing using the rbcL marker gene . protocols.io dx.doi.org/10.17504/protocols.io.bd94i98w Copy
Authors: David Eccles
Group: High molecular weight DNA extraction from all kingdoms
Summary: This protocol is for a semi-manual method for read demultiplexing, as used after my presentation Sequencing DNA with Linux Cores and Nanopores to work out the number of reads captured by different barcodes.Input: reads as a FASTQ file, barcode sequences as a FASTA fileOutput: reads split into single FASTQ files per target [barcode]Note: barcode / adapter sequences are not trimmed by this protocol
Proper citation: David Eccles 2019. Demultiplexing Nanopore reads with LAST. protocols.io dx.doi.org/10.17504/protocols.io.xj3fkqn Copy
Authors: Angel Justiz-Vaillant
Summary: A protein that combines the binding capacity of SpA and SpG is not comercially available. It can be easy created in the laboratory by combining these two immunoglobulin-binding proteins to horseradish peroxidase by the periodate method [1]. However, a mixture of SpA and SpG could have the same effect as universal reagent in immunodetection. 1. Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175
Proper citation: Angel Justiz-Vaillant 2020. Universal Immunoblot analysis for investigating Protein-AG (SpAG)-binding to avian and mammalian immunoglobulins.. protocols.io dx.doi.org/10.17504/protocols.io.bjseknbe Copy
Authors: Ben Tully
Group: ECOGEO
Summary: mothur is a bioinformatics tool for analyzing 16S rRNA gene sequences, which can be used to process data generated by Sanger, PacBio, IonTorrent, 454, and Illumina (MiSeq/HiSeq).
Proper citation: Ben Tully 2016. ECOGEO 'Omics Training: 3.1 Amplicon Analysis - Mothur. protocols.io dx.doi.org/10.17504/protocols.io.fi4bkgw Copy
Authors: Sam Mugford, Roland Wouters, Thomas C Mathers, Saskia Hogenhout
Summary: Genomic studies of natural populations frequently benefit by having data from single individuals. This approach has a number of advantages: it avoids the risk of accidently pooling together individuals of closely related species, and it enables better resolution of haplotypes by separating non-identical individuals. However, especially in the case of very small invertebrates, recovering sufficient nucleic acid from individual samples can be challenging. Here we present a method suitable for extraction of DNA from single individual aphids (weighing approximately 0.5mg). We are able to recover 100’s of ng of high-quality DNA suitable for whole-genome sequencing using short-read technologies. DNA extraction using Cetrimonium bromide (CTAB) is a tried and tested method, and the protocol presented here is adapted from that of Marzachì et al. (1998). We present some modifications to optimize this popular method to maximize yield and quality from small single insect samples.We have used this method across a range of aphid species, and also other sap-sucking hemipteran insects. It has enabled sequencing and de-novo assembly of a range of species from single individuals collected from the field, and also resequencing of individuals of the model aphid Myzus persicae. This is compatible with the method of ambient storage of dehydrated filed collected samples we describe in Wouters et al. (2020).Marzachì, C, Veratti, F, & Bosco, D (1998). Direct PCR detection of phytoplasmas in experimentally infected insects. Annals of Applied Biology, 133(1), 45-54.Wouters, R; Mugford S, & Hogenhout, S (2020). Ambient sample storage system of field-collected insect samples for genomics. protocols.io dx.doi.org/10.17504/protocols.io.bgxnjxme
Proper citation: Sam Mugford, Roland Wouters, Thomas C Mathers, Saskia Hogenhout 2020. High quality DNA extraction from very small individual insects. protocols.io dx.doi.org/10.17504/protocols.io.bg6wjzfe Copy
Authors: Gina Filloramo
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Summary: Successful transformation of Phaeodactylum tricornutum (CMP632) by electroporation was achieved based on a modified version of Zhang & Hu (2014).
Proper citation: Gina Filloramo 2018. Transformation of Phaeodactylum tricornutum by electroporation. protocols.io dx.doi.org/10.17504/protocols.io.s4cegsw Copy
Authors: Alexandra Netter-Glangeaud
Group: StressMarq
Proper citation: Alexandra Netter-Glangeaud 2019. Tau Thioflavin T Assay. protocols.io dx.doi.org/10.17504/protocols.io.wxgffjw Copy
Authors: Addgene The Nonprofit Plasmid Repository
Summary: This protocol describes plasmid modification by annealed oligo cloning. To see the full abstract and additional resources, please visit the Addgene protocol page.
Proper citation: Addgene The Nonprofit Plasmid Repository 2019. Plasmid Modification by Annealed Oligo Cloning. protocols.io dx.doi.org/10.17504/protocols.io.47tgznn Copy
Authors: Daniel Vaulot
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff
Summary: Protocol to fix marine samples for flow cytometry sorting of phytoplankton. Fix at least 2 samples per depth sampled and at least 6 to 10 depths per vertical profile.Back to the laboratory, pico and nano-plankton populations can be sorted and used for clone library construction or metabarcoding with Next Generation Sequencing (e.g. Illumina). This was successfully tested on samples from a cruise off Brazil (unpublished data)ReferenceRibeiro C., Lopes A., Marie D., Vaulot D. Unpublished data
Proper citation: Daniel Vaulot 2015. Fixation of marine samples for flow cytometry sorting. protocols.io dx.doi.org/10.17504/protocols.io.d2x8fm Copy
Authors: Daniel Richter
Group: Ecology of Marine Plankton (ECOMAP) team - Roscoff, Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Daniel Richter 2017. Modified Qiagen PCR purification (no gel extraction) with MinElute spin column.. protocols.io dx.doi.org/10.17504/protocols.io.ivece3e Copy
Authors: Nus Igem
Proper citation: Nus Igem 2019. Preparation of Chemically Competent Cells. protocols.io dx.doi.org/10.17504/protocols.io.7pnhmme Copy
Authors: Maximilian Dietsch, Vera Wewer
Group: Axmann Lab, CyanoWorld
Summary: In this protocol, the extraction of naturally occurring as well as heterologously synthesized triterpenes in Synechocystis and R. capsulatus are described
Proper citation: Maximilian Dietsch, Vera Wewer 2018. Triterpene extraction protocol from Synechocystis sp. PCC6803. protocols.io dx.doi.org/10.17504/protocols.io.mspc6dn Copy
Authors: avinash.kale
Group: UM-DAE Centre for Excellence in Basic Sciences
Proper citation: avinash.kale 2020. DNA extraction and Sequencing. protocols.io dx.doi.org/10.17504/protocols.io.bjdxki7n Copy
Authors: Jakob H. Andersen
Proper citation: Jakob H. Andersen 2017. Does Perineural Clonidine Prolong the Duration of an Adductor Canal Block When Controlling for Possible Systemic effects? - A Randomized Paired Trial in Healthy Volunteers. protocols.io dx.doi.org/10.17504/protocols.io.hsnb6de Copy
Authors: Tzu-yen Huang
Proper citation: Tzu-yen Huang 2020. Meta-analysis of scope harvesting radial artery. protocols.io dx.doi.org/10.17504/protocols.io.bhrdj526 Copy
Authors: Jeff Strohm, Robert Hanner, Richard J Heck
Summary: This is a collection of methods from [tentative citation]:
Jeff Strohm, Robert Hanner, Richard J Heck. (2018) A metabarcoding perspective on soil biodiversity under different tillage treatments. Submitted to Metabarcoding and Metagenomics
Proper citation: Jeff Strohm, Robert Hanner, Richard J Heck 2018. Methods from: The influence of agricultural tillage practices on soil biodiversity. protocols.io dx.doi.org/10.17504/protocols.io.efsbbne Copy
Authors: Denville Scientific
Group: Denville Scientific, Inc.
Summary: The SpinSmart PCR purification and gel extraction technologies utilize a lysis buffer containing chaotropic salts that allow DNA to bind to a silica membrane. Binding buffer PCR 1 is added to a PCR reaction or agarose gel slice; the mixture is subsequently loaded directly onto SpinSmart PCR Columns. Salts, enzymes, and other soluble components are washed away with ethanolic PCR 2 Wash buffer. Purified DNA is eluted using PCR 3 Elution buffer (5 mM Tris/HCl, pH 8.5). Please follow one of the protocols below, depending on whether you are doing a DNA gel extraction or PCR purification.
Proper citation: Denville Scientific 2016. SpinSmart PCR Purification and Gel Purification Procedures. protocols.io dx.doi.org/10.17504/protocols.io.gr6bv9e Copy
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