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Authors: Matema Imakumbili
Summary: This protocol describes how foliar and pesticide solutions can be made and applied to growing crops. Foliar solutions (foliar fertilisers) can be either made using water soluble fertilisers (e.g. urea), inorganic salts (e.g. KCl, K2SO4) or by using readymade manufactured concentrated foliar solutions that could be in the form of chelated compounds of metallic ions. Their application rates are often simply stated as a percentage of the nutrient to be applied in solution form e.g. a 1 - 2% Zn foliar fertiliser solution. This protocol describes how to determine the amount of chemical material needed to make a specified concentration of a foliar solution mixture using various sources. Although not always stated, except on manufactured foliar solutions, foliar fertilisers should be ideally applied over a specified area to ensure that plants receive a sufficient volume of foliar solution. This protocol thus also describes how to make a foliar solution taking into consideration the area over which they shall be applied. Like readymade manufactured foliar solutions, pesticides also have instructions on how to make and apply their solutions over a specified area. How pesticide solutions should be made and applied according to specified instructions will hence also be shown. Instructions for making foliar and pesticide solutions can be a little tricky to follow if you are not accustomed to the technical language. This protocol has been designed to give people some help with this.
Proper citation: Matema Imakumbili 2020. Making and applying foliar fertiliser and pesticide solutions. protocols.io dx.doi.org/10.17504/protocols.io.bbswinfe Copy
Authors: Roey Angel, Eva Petrova, Ana Lara
Group: SoWa RI Anaerobic and Molecular Microbiology (public)
Summary: The following protocol describes how to perform an RNA-Stable Isotope Probing experiment. The scope of this protocol only covers the parts involving separating labelled RNA from unlabelled RNA using ultracentrifugation in a caesium trifluoroacetate density gradient and downstream quantification to evaluate whether the labelling and separation of the RNA were successful. Total RNA should be extracted from an environmental sample or an enrichment culture that was incubated with an isotopically-labelled substrate. Labelling can be of the carbon, oxygen or nitrogen in the RNA (or any combination of the 3). For environmental samples, we recommend extracting RNA using our protocol Total Nucleic Acids Extraction from Soil and purifying it using the Purification of RNA from Crude NA Extract protocol. This protocol is based on the following papers: Whiteley et al. (2007); Dumont et al. (2011); Angel and Conrad (2013). For a comprehensive discussion on how to design a SIP experiment and how to analyse the resulting data, we recommend referring to the recent book on the subject: Stable Isotope Probing: Methods and Protocols, especially chapters: 1-3 and 9-18.
Proper citation: Roey Angel, Eva Petrova, Ana Lara 2021. RNA-Stable Isotope Probing. protocols.io dx.doi.org/10.17504/protocols.io.bsbxnapn Copy
Authors: Gabriela Canalli Kretzschmar, Luana Caroline Oliveira, Angelica Beate Winter Boldt
Summary: This is a simple PCR-SSP for amplification of the rs6656401, located in intron 4 (CR1 gene).
Proper citation: Gabriela Canalli Kretzschmar, Luana Caroline Oliveira, Angelica Beate Winter Boldt 2018. Simple PCR-SSP- rs6656401-In4 - CR1. protocols.io dx.doi.org/10.17504/protocols.io.p44dqyw Copy
Authors: Rebecca Ann Helmer, Gurvinder Kaur, Lisa Ann Smith, Beverly S. Chilton
Summary: The helicase-like transcription factor (HLTF) gene – a tumor suppressor in human colorectal cancer (CRC) - is regulated by alternative splicing and promoter hypermethylation. The detection of hypermethylated HLTF DNA in fecal occult blood tests is an indicator of disease recurrence and poor survival. Hltf-deficiency in the ApcMin/+ mouse strain increased the formation of intestinal adenocarcinoma with a high incidence of gross chromosomal instabilities. To investigate Hltf-deletion effects in CRC without cross-breeding into a tumorigenic strain, Hltf-deletion was studied in mice treated with the carcinogen azoxymethane (AOM) and the proinflammatory agent dextran sodium sulfate (DSS). Hltf-deletion resulted in weight loss beginning at treatment week 6, and poor survival (Kaplan-Meier survival plot). Hltf-deletion increased tumor multiplicity compared to controls, and dramatically shifted the topographic distribution of lesions into the rectum. Differential isoform expression analysis of lesions from control mice revealed both the truncated isoform that lacks a DNA-repair domain and the full length isoform capable of DNA damage repair are present (3:1.8 ratio) during adenocarcinoma formation. iPathwayGuide identified 51 dynamically regulated genes of 10,967 total genes with measured expression. Oxidative Phosphorylation (Kegg: 00190), the top biological pathway perturbed by Hltf-deletion, resulted from increased transcription of Atp5e, Cox7c, Uqcr11, Ndufa4 and Ndufb6 genes, concomitant with increased endogenous levels of ATP (p=0.0176). Upregulation of gene expression, as validated with qRT-PCR, was accompanied by a stable mtDNA/nDNA ratio. This is the first study to show Hltf-deletion in an inflammation-associated CRC model elevates mitochondrial bioenergetics. The distal shift in tumorigenesis indicates the detection of hypermethylated HLTF DNA in stool samples might be a prognostic biomarker for distal (left-sided) CRC in patients with inflammatory bowel disease.
Proper citation: Rebecca Ann Helmer, Gurvinder Kaur, Lisa Ann Smith, Beverly S. Chilton 2019. Helicase-like transcription factor (Hltf) gene-deletion promotes oxidative phosphorylation (OXPHOS) in colorectal tumors of AOM/DSS-treated mice. protocols.io dx.doi.org/10.17504/protocols.io.4argsd6 Copy
Authors: Ricardo Quiteres, Alvaro Crevenna, Zach Hensel, Federico Herrera
Summary: Live-cell images of U251 cells were acquired at Instituto Gulbenkian de Ciência on a commercial widefield Nikon High Content Screening microscope, equipped with a 100x/1.45 plan-apo oil-immer- sion objective and an Andor Zyla 4.2 Scientific complementary metal-oxide-semiconductor (sCMOS) camera. To image eGFP/Venus–fused proteins, a 470 nm laser line, and GFP fluorescence filtersets were used and for the imaging of HaloTag-fused proteins, a 635 nm laser line and Cy5 fluorescent filtersets were used. An exposure time of 80 ms was used and the camera readout bandwidth was set to 200MHz.The resulting pixel size was 65 nm using a 1024x1024 pixel as field of view. All acquisition were done at room temperature.The microscope, cameras, and hardware were controlled through Nikon Elements software. Images were analyzed by means of the ImageJ free software.
Single-molecule imaging and Super-Resolution Radial Fluctuations (SRRF) imaging were performed on a Leica DMI6000 inverted microscope using a 100x/1.46 a-plan apochromat oil immersion objective. A 561 nm excitation laser (Coherent Sapphire) was set to 50 mW resulting in an effective power density of ~2.3kW/cm2. The laser beam was passed through a custom filter cube (Chroma Technology) with a zet405/561x excitation filter, a zt405/561/657rpc-uf2 dichroic beam splitter, and an et610/75m emission filter. Fluorescent light was imaged on an Evolve512 electron multiplying charge-coupled device (EM-CCD) camera (Photometrics) after additional 1.6x magnification. An EM gain of 300 was used and the camera read out band width was set to 10 MHz. The resulting pixel size was 100 nm using a 512 x 512 pixel as field of view. The incubation chamber was maintained at 37°C. The microscope , cameras, and hardware were controlled through MetaMorph software (Molecular Devices). For single-molecule experiments and for each cell, 1000 frames were acquired at 33 ms exposure time with no interval between frames. No photoactivation was necessary as spontaneous photoactivation of PA-JF549 combined with photobleaching by 561 nm illumination gave a reasonable density of single molecules.The Trackmate ImageJ plugin was used to detect, fit and track individual GFAP molecules in living cells (A-C). For the detection of GFAP molecules, we selected a LoG detector and an estimated blob diameter of 0,4 mm and a threshold of 1500. For the tracking, we used a simple LAP tracker with a linking maximum distance of 0,5 mm, a gap-closing maximum distance of 0,5 mm and a gap-closing max frame gapof0.The average diffusion of GFAP molecules and its fractions, per individual cell, were calculated by fitting the SpotOn 2-state(bound-free) kinetic model (0,05mm2/s as maximum Dbound and 0,02 mm2/s as minimum Dfree) to the distribution of translocations for individual molecules, obtained with TrackMate. For SRRF imaging, 100 frames were acquired for each cell. SRRF images were generated by running the NanoJ-SRRF ImageJ plugin on groups of 100 diffraction-limited images using the default settings.
Proper citation: Ricardo Quiteres, Alvaro Crevenna, Zach Hensel, Federico Herrera 2021. Microscopy. protocols.io dx.doi.org/10.17504/protocols.io.77ghrjw Copy
Authors: Andrew Potter
Group: Human Cell Atlas Method Development Community
Summary: Protocol for adult (8-10 week) mouse kidney dissociation performed on ice to reduce artifact gene expression. The first layer, consisting of collagenase digestion, breaks down the tissue and releases some cells and glomeruli and tubules. The second layer consists of bacillus licheniformis digestion for 15 min. augmented with a thermomixer at 1400 RPM and passaging with a 27 gauge needle. The second layer is meant to thoroughly break up remaining tubules and glomeruli, releasing cells such as podocytes. The final yield is 250K cells from 18 mg tissue with 98% viability, approximately 14,000 cells/mg tissue. Approximately 1% of released cells are podocytes (visualized using kidneys from MAFB-GFP+ mice using a hemocytometer).
Proper citation: Andrew Potter 2018. Adult mouse kidney dissociation (on ice). protocols.io dx.doi.org/10.17504/protocols.io.rynd7ve Copy
Authors: Alle Lie
Group: Protist Research to Optimize Tools in Genetics (PROT-G), Caron Lab - Protistan Ecology
Proper citation: Alle Lie 2016. Algal culture harvest and RNA extraction for RNA-Seq. protocols.io dx.doi.org/10.17504/protocols.io.ewjbfcn Copy
Authors: Roscoff Culture Collection
Group: Roscoff Culture Collection
Summary: Medium to grow phytoplankton species in particular dinoflagellates which often require soil extract.
Proper citation: Roscoff Culture Collection 2019. f/2 medium with soil extract. protocols.io dx.doi.org/10.17504/protocols.io.s29egh6 Copy
Authors: Anthony Bellantuono
Group: Aiptasia-Symbiodiniaceae Model System
Summary: This protocol lyses symbiodinium cells with heat and pellets debris, producing material suitable as crude DNA template for robust PCRs. It is convenient for rapid strain identification from liquid cultures and from Symbiodinium-hosting Aiptasia anemones.
Proper citation: Anthony Bellantuono 2018. Symbiodinium / Aiptasia cell pop (crude lysis for PCR template). protocols.io dx.doi.org/10.17504/protocols.io.k2bcyan Copy
Authors: Lira Mamanova
Proper citation: Lira Mamanova 2019. Tissue dissociation protocol for Pan Immune Project Tissue. protocols.io dx.doi.org/10.17504/protocols.io.ymzfu76 Copy
Authors: Dylan P. Smith, Kabir G. Peay, Gail Ackermann, Amy Apprill, Markus Bauer, Donna Berg-Lyons, Jason Betley, T. D. Bruns, J. Greg Caporaso, Noah Fierer, Louise Fraser, Jed A. Fuhrman, M. Gardes, Jack A. Gilbert, Niall Gormley, Greg Humphrey, James Huntley, Janet K. Jansson, Rob Knight, Chris L. Lauber, S. Lee, Sarah M. Owens, Alma E. Parada, Geoff Smith, J. Taylor, Luke Thompson, Willam A. Walters, T. J. White
Group: Earth Microbiome Project
Summary: The ITS protocol detailed here is designed to amplify fungal microbial eukaryotic lineages using paired-end community sequencing on the Illumina platform with primers ITS1f-ITS2 (EMP.ITSkabir).
Proper citation: Dylan P. Smith, Kabir G. Peay, Gail Ackermann, Amy Apprill, Markus Bauer, Donna Berg-Lyons, Jason Betley, T. D. Bruns, J. Greg Caporaso, Noah Fierer, Louise Fraser, Jed A. Fuhrman, M. Gardes, Jack A. Gilbert, Niall Gormley, Greg Humphrey, James Huntley, Janet K. Jansson, Rob Knight, Chris L. Lauber, S. Lee, Sarah M. Owens, Alma E. Parada, Geoff Smith, J. Taylor, Luke Thompson, Willam A. Walters, T. J. White 2018. EMP ITS Illumina Amplicon Protocol. protocols.io dx.doi.org/10.17504/protocols.io.pa7dihn Copy
Authors: Zilma Reis, Juliano de Souza Gaspar, Sara Oliveira Elias, Regina Amelia Lopes Pessoa De Aguiar
Summary: This protocol presents an algorithm for gestational age assessment when a reliable last menstrual period or an obstetric ultrasound are available in the birth scenario. A software was developed to automatically process data entries into the best estimative of the gestational age at birth. It shall be used by the multicenter team of researchers, duly trained in accordance with the Good Clinical Practice Protocol, during the enrollment of newborns. Also, this protocol is complementary documentation for the scientific publications related to the clinical trials:"Prematurity detection evaluating interaction between the skin of the newborn and light: protocol for the preemie-test multicentre clinical trial in Brazilian hospitals to validate a new medical device"; Register number RBR-3f5bm5."Premature or small for gestational age? International multicenter trial protocol for classification of the low birth weight newborn through the optical properties of the skin"; Register number RBR33rnjf.
Proper citation: Zilma Reis, Juliano de Souza Gaspar, Sara Oliveira Elias, Regina Amelia Lopes Pessoa De Aguiar 2020. Algorithm for gestational age assessment at birth. protocols.io dx.doi.org/10.17504/protocols.io.bawbifan Copy
Authors: John-Sebastian Eden, Eby Sim
Group: Coronavirus Method Development Community
Summary: This protocol describes methods to sequence SARS-CoV-2 with pooled amplicons (14 x 2.5kb) using Illumina Platforms.
Proper citation: John-Sebastian Eden, Eby Sim 2020. SARS-CoV-2 Genome Sequencing Using Long Pooled Amplicons on Illumina Platforms. protocols.io dx.doi.org/10.17504/protocols.io.befyjbpw Copy
Authors: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen
Group: VERVE Net, Suttle Laboratory of Marine Molecular Microbiology and Virology
Summary: For use in "Isolation of cyanophages by plaque assyays"
Proper citation: Mathias Middelboe, Amy M. Chan, and Sif K. Bertelsen 2016. Top agarose. protocols.io dx.doi.org/10.17504/protocols.io.dqh5t5 Copy
Authors: Thomas Oliver
Proper citation: Thomas Oliver 2016. Coral TRIZOL RNA Extraction. protocols.io dx.doi.org/10.17504/protocols.io.egibbue Copy
Authors: Shaohua Huang, Yi Xing, Lijuan Zhu, Ke Zhang
Summary: Background:Fixed-combination (FC) therapy is used in primary open-angle glaucoma (POAG) and ocular hypertension (OHT) patients who require more than one medication to reach their target intraocular pressure (IOP). Currently, there are several FC therapies available for the treatment of glaucoma. The FC of latanoprost/timolol (LTFC) is a commonly used. We will conduct a systematic review to compare the IOP-lowering effects of LTFC with other FCs for patients with POAG and OHT.Materials and methods: We will search PubMed, EMBASE, the Cochrane Library, and Web of Sciencefor randomized-controlled clinical trials and cross-over studies. The outcomes are mean IOP and IOP fluctuation after one month of treatment. Meta-analysis will be carried out using RevMan (version 5.1) software. After conducting meta-analyses, we will rate the quality of each meta-analysis as high, moderate, low, or very low using the “GRADE” system.
Proper citation: Shaohua Huang, Yi Xing, Lijuan Zhu, Ke Zhang 2020. Protocol for the systematic review of the efficacy of the fixed combination of latanoprost and timolol versus other fixed combinations for primary open-angle glaucoma and ocular hypertension. protocols.io dx.doi.org/10.17504/protocols.io.bbsfinbn Copy
Authors: Ken Christensen
Summary: Working alone in the laboratory should be avoided; however, trained personnel can work alone in the laboratory if they adhere to the following Standard Operating Procedure (SOP). Students in training (during at least the 1st semester of laboratory work) cannot work alone in the laboratory.
Proper citation: Ken Christensen 2020. Working Alone in the Lab . protocols.io dx.doi.org/10.17504/protocols.io.beiajcae Copy
Authors: Bonnie Hurwitz
Group: Metafunc Course 2017
Summary: Let's start to download the fastq files for your project.
Proper citation: Bonnie Hurwitz 2017. Week 2. protocols.io dx.doi.org/10.17504/protocols.io.jwccpaw Copy
Authors: Lijun Zhang
Proper citation: Lijun Zhang 2019. Supplement Figure S3. protocols.io dx.doi.org/10.17504/protocols.io.yfgftjw Copy
Authors: Ran Zhou
Group: Human Cell Atlas Method Development Community, Helmsley project_Basu lab
Summary: The protocol is adapted from Fujii's and Smillies's reports for single cell transcriptome analysis from human intestines. It provides details on acquirement of single cell suspension from epithelium and lamina propria. This methods is modified to generate appropriate meterials from patient's intestinal biopsies for sinlge-cell transcriptome and genomic applications.
Proper citation: Ran Zhou 2020. Preparation of single cell suspensions from human intestinal biopsies for single cell genomics applications. protocols.io dx.doi.org/10.17504/protocols.io.bde3i3gn Copy
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