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Authors: Carly Martin, Ashley Feirrera, Evan Macosko
Group: Human Cell Atlas Method Development Community
Summary: Protocol for extraction of nuclei from frozen tissue in preparation for single-nuclei sequencing (droplet-based/10X). This protocol is based strongly on a similar extraction protocol from the McCarroll lab.
Proper citation: Carly Martin, Ashley Feirrera, Evan Macosko 2019. Frozen Tissue Nuclei Extraction. protocols.io dx.doi.org/10.17504/protocols.io.2frgbm6 Copy
Authors: Jia Ren Lin
Group: Laboratory of Systems Pharmacology, NCIHTAN
Summary: The hematoxylin and eosin (H&E) stain is the standard used for microscopic examination of fixed tissue specimens. This procedure establishes a consistent process for the manual preparation of H&E-stained slides from FFPE tissue specimens. H&E staining is compatible with, and frequently performed after tissue-based cyclic immunofluorescence (t-CyCIF), enabling conventional histopathology review to be combined with fluorescence imaging.
Proper citation: Jia Ren Lin 2021. Manual H&E Staining. protocols.io dx.doi.org/10.17504/protocols.io.bsi8nchw Copy
Authors: Ian Mackay, Judy Northill
Group: Public Health Virology, Forensic and Scientific Services
Summary: This protocol aims to amplify enterovirus (EV) A71 viruses but not other viruses.This protocol was designed by us.The oligonucleotides target the 5'UTR noncoding region. This is a qualitative test for investigating EV-A71 infection of humans.The test has identified both historical EV-A71 strains and contemporary strains and has confirmed these using subgenomic sequencing of partial 5'UTR-VP2 and partial VP1 sequencing. Culture of the virus is not required as this assay is capable of detecting EV-A71 direct from extracted clinical samples.
Proper citation: Ian Mackay, Judy Northill 2019. Enterovirus (EV) A71 real-time RT-PCR (EV-A71-TM2018). protocols.io dx.doi.org/10.17504/protocols.io.xb3fiqn Copy
Authors: Georgios I Laliotis, Philip N. Tsichlis
Proper citation: Georgios I Laliotis, Philip N. Tsichlis 2020. Effective identification of RNA-binding proteins using RNA Immunoprecipitation. protocols.io dx.doi.org/10.17504/protocols.io.bjpbkmin Copy
Authors: Gustaf Degen
Proper citation: Gustaf Degen 2019. Transformation of E.coli with pGem-T Easy. protocols.io dx.doi.org/10.17504/protocols.io.hqab5se Copy
Authors: Rowan Mclachlan, Agus Munoz-Garcia, Andrea Grottoli
Group: Coral Bleaching RCN protocols
Summary: This method is adapted and updated from methods originally published in Grottoli et al. (2004) and is based on the original methods of Folch & Stanley (1957), and Bligh & Dyer (1959). There are five parts to extracting lipids from ground corals: 1) grind and sub-sample the coral and store at -80 °C until ready to extract, 2) freeze-dry the sample, 3) extract the lipids from the freeze-dried samples, 4) standardize the lipid concentration to ash-free dry weight (AFDW), and 5) resuspend the extracted lipid for long-term storage and possible later analysis of lipid classes or isotopes. The lipid extraction procedure must be conducted in a fume hood with the sash as low as possible with the researcher wearing protective eyewear, gloves, and lab coat at all times.Important considerations regarding lipid analysis were gained from reading Chapter 1.3 “Lipid extraction, storage, and sample handling” from the textbook Lipid Analysis by Christie (2003). This method was originally developed by Andréa Grottoli and refined by Rowan McLachlan (06-11-18) with the guidance of Dr. Agus Muñoz-Garcia at The Ohio State University. This protocol was written by Rowan McLachlan (03-12-2020).ReferencesBligh, E. G., & Dyer, W. J. (1959). Canadian Journal of Biochemistry and Physiology. Canadian Journal of Biochemistry and Physiology, 37(8), 911–917. Christie, W. W. (2003). Lipid Analysis: Vol. 15, Isolation, Separation, Identification and Structural Analysis of Lipids. Current Opinion Biotechnology (3rd ed., Vol. 4). Oily Press Ltd, UK.Folch, J., Lees, M., & Stanley, G. H. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, 497-509.Grottoli A. G., Rodrigues L. J., & Juarez C. (2004). Lipids and stable carbon isotopes in two species of Hawaiian corals, Porites compressa and Montipora verrucosa, following a bleaching event. Marine Biology, 145: 621–631.Christie, W. W. (2003). Lipid Analysis: Vol. 15, Isolation, Separation, Identification and Structural Analysis of Lipids. Current Opinion Biotechnology (3rd ed., Vol. 4). Oily Press Ltd, UK.Folch, J., Lees, M., & Stanley, G. H. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, 497-509.Grottoli A. G., Rodrigues L. J., & Juarez C. (2004). Lipids and stable carbon isotopes in two species of Hawaiian corals, Porites compressa and Montipora verrucosa, following a bleaching event. Marine Biology, 145: 621–631.Folch, J., Lees, M., & Stanley, G. H. S. (1957). A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry, 226, 497-509.Grottoli A. G., Rodrigues L. J., & Juarez C. (2004). Lipids and stable carbon isotopes in two species of Hawaiian corals, Porites compressa and Montipora verrucosa, following a bleaching event. Marine Biology, 145: 621–631.
Proper citation: Rowan Mclachlan, Agus Munoz-Garcia, Andrea Grottoli 2020. Extraction of Total Soluble Lipid from Ground Coral Samples. protocols.io dx.doi.org/10.17504/protocols.io.bc4qiyvw Copy
Authors: Molly Miranda
Group: Genomes to Natural Products Network
Summary: This method describes magnetic particle based DNA purification.
Proper citation: Molly Miranda 2017. Magnetic particle based DNA purification. protocols.io dx.doi.org/10.17504/protocols.io.g4abyse Copy
Authors: Joshua Timmons
Group: Northeastern iGEM 2015
Summary: Separates molecules based on size.
Proper citation: Joshua Timmons 2015. Gel Electrophoresis. protocols.io dx.doi.org/10.17504/protocols.io.dqa5sd Copy
Authors: Gomard Yann, Lagadec Erwan and Tortosa Pablo
Summary: This protocol is adapted from that detailed in the International Course on Laboratory methods for the Diagnosis of Leptospirosis, by Dr. R.A. Hartskeerl, Dr. H.L. Smits, Mr. H. Korver, Dr. M.G.A. Goris and Dr. W.J. Terpstra from the Leptospirosis Reference Center in Amsterdam, the Netherlands. It is routinely used at UMR PIMIT, St Denis de La Réunion, France, for the isolation of pathogenic Leptospira from wild animals (terrestrial small mammals, bats and dogs). For Leptospira isolation from urine, go directly to step 10.
Proper citation: Gomard Yann, Lagadec Erwan and Tortosa Pablo 2017. Detailed protocol for Leptospira isolation from animal samples. protocols.io dx.doi.org/10.17504/protocols.io.ifccbiw Copy
Authors: Robert Hooftman
Group: iGEM Wageningen 2019
Summary: In this protocol, the preparation and running an acrylamide gel is explained. Using this gel, proteins with a size of >15 kDa can be visualized. For the visualization of proteins with a size of 15 -30 kDa, a 12% acrylamide gel can be used. For the visualization of proteins with a size of 30-50 kDa, a 10% acrylamide gel can be used.
Proper citation: Robert Hooftman 2019. Making and running an acrylamide protein gel. protocols.io dx.doi.org/10.17504/protocols.io.7syhnfw Copy
Authors: Marcelo Josende, Silvana Manske Nunes, Larissa Müller, Marlize Ferreira Cravo, José Marìa Monserrat, Juliane Ventura Lima
Summary: This protocol is part of the entitled paper 'Circular estimate method (CEM) - A simple method to estimate Caenorhabditis elegans culture densities in liquid medium.' DOI: 10.1186/s12575-018-0089-2, published at Biological Procedures Online (2019), and describes the step-by-step procedures for the achievement of accurate and reliable estimates with the extremely close relationship to the real nematode counting. It can be used on different nematode species and adapted to different laboratory conditions.
Proper citation: Marcelo Josende, Silvana Manske Nunes, Larissa Müller, Marlize Ferreira Cravo, José Marìa Monserrat, Juliane Ventura Lima 2019. CEM (Circular Estimate Method) – Step-by-step Protocol. protocols.io dx.doi.org/10.17504/protocols.io.xhmfj46 Copy
Authors: Damiano Menin, Angela Costabile, Flaviana Tenuta, Harriet Oster, Marco Dondi
Summary: In these two studies, we examined the temporal dynamics of yawning and non-yawn mouth openings in preterm neonates comparable in developmental level to fetuses observed in ultrasound studies (about 31 weeks postmenstrual age, PMA). In Study 1 we tested the reliability and construct validity of the only quantitative measure for identifying fetal yawns in the literature, by comparing its scores with a more detailed behavioral coding system (The System for Coding Perinatal Behavior, SCPB) adapted from the comprehensive, anatomically based Facial Action Coding System for Infants and Young Children (Baby FACS). In Study 2 we developed and tested a new machine learning system based on support vector machines (SVM) for identifying yawns.
Proper citation: Damiano Menin, Angela Costabile, Flaviana Tenuta, Harriet Oster, Marco Dondi 2019. Identify fetal yawns based on temporal dynamics of mouth opening. . protocols.io dx.doi.org/10.17504/protocols.io.739hqr6 Copy
Authors: Hansheng Zhao
Proper citation: Hansheng Zhao 2018. ALLPATHS-LG Genome Assembly and Hi-C mapping strategy. protocols.io dx.doi.org/10.17504/protocols.io.phmdj46 Copy
Authors: Bao Thai
Group: Stephen Floor Lab
Proper citation: Bao Thai 2018. WB SDS loading buffer (5X). protocols.io dx.doi.org/10.17504/protocols.io.mn9c5h6 Copy
Authors: Hazel Rogers, Laura Letchford, Sara Vieira, Maria Garcia-Casado, Mya Fekry-Troll, Charlotte Beaver, Rachel Nelson, Hayley Francies, Mathew Garnett
Group: Cellular Generation and Phenotyping
Summary: This protocol describes the passaging of organoid cultures. It has been developed by the organoid derivation team within the Cellular Generation and Phenotyping Group at the Wellcome Sanger Institute. The team has extensive experience passaging and expanding organoid models. The method described has mainly been used for the passaging cancer organoids with successful propagation of organoids derived from colon, pancreas and oesophageal tumours.
Proper citation: Hazel Rogers, Laura Letchford, Sara Vieira, Maria Garcia-Casado, Mya Fekry-Troll, Charlotte Beaver, Rachel Nelson, Hayley Francies, Mathew Garnett 2020. Passaging cancer organoid cultures. protocols.io dx.doi.org/10.17504/protocols.io.bfe3jjgn Copy
Authors: Derya Cokal, Vitor Zimmerer, Douglas Turkington, Nicol Ferrier, Rosemary Varley, Stuart Watson, Wolfram Hinzen
Summary: Everyday speech is produced with an intricate timing pattern and rhythm. Speech units follow each other with short interleaving pauses, which can be either bridged by fillers (erm, ah) or empty. Through their syntactic positions, pauses connect to the thoughts expressed. We investigated whether disturbances of thought in schizophrenia are manifest in patterns at this level of linguistic organization, whether these are seen in first degree relatives (FDR) and how specific they are to formal thought disorder (FTD). Spontaneous speech from 15 participants without FTD (SZ-FTD), 15 with FTD (SZ+FTD), 15 FDRs and 15 neurotypical controls (NC) was obtained from a storytelling task and rated for pauses subclassified by syntactic position and duration. SZ-FTD produced significantly more unfilled pauses than NC in utterance-initial positions and before embedded clauses. Unfilled pauses occurring withinclausal units did not distinguish any groups. SZ-FTD also differed from SZ+FTD in producing significantly more pauses before embedded clauses. SZ+FTD differed from NC and FDR only in ‘long’ (between 1 and 3 seconds) utterance-initial pauses. FDRs produced significantly fewer fillers than NC. Results reveal that the temporal organization of speech is an important window on disturbances of the thought process and how these relate to language.
Proper citation: Derya Cokal, Vitor Zimmerer, Douglas Turkington, Nicol Ferrier, Rosemary Varley, Stuart Watson, Wolfram Hinzen 2019. Disturbing the rhythm of thought: speech pausing patterns in schizophrenia, with and without formal thought disorder. protocols.io dx.doi.org/10.17504/protocols.io.zqrf5v6 Copy
Authors: Aicha Melouane, Mayumi Yoshioka, Jonny St-Amand
Group: Molecular Neuroplasticity
Summary: Secreted protein, acidic and rich in cysteine (SPARC) is differentially associated with cell proliferation and extracellular matrix (ECM) assembly. We show here the effect of exogenous SPARC inhibition/induction on ECM and mitochondrial proteins expression and on the differentiation of C2C12 cells. The cells were cultured in growth medium (GM) supplemented with different experimental conditions. The differentiation of myoblasts was studied for 5 days, the expressions of ECM and mitochondrial proteins were measured and the formation of the myotubes was quantified after exogenous induction/inhibition of SPARC. The results indicate that the addition of recombinant SPARC protein (rSPARC) in cell culture medium increased the differentiation of C2C12 myoblasts and myogenin expression during the myotube formation. However, the treatment with antibody specific for SPARC (anti-SPARC) prevented the differentiation and decreased myogenin expression. The induction of SPARC in the proliferating and differentiating C2C12 cells increased collagen 1a1 protein expression, whereas the inhibition decreased it. The effects on fibronectin protein expression were opposite. Furthermore, the addition of rSPARC in C2C12 myoblast increased the expression of mitochondrial proteins, ubiquinol-cytochrome c reductase core protein II (UQCRC2) and succinate dehydrogenase iron-sulfur subunit (SDHB), whereas the anti-SPARC decreased them. During the differentiation, only the anti-SPARC had the effects on mitochondrial proteins, NADH dehydrogenase ubiquinone 1 beta subcomplex subunit 8 (NADHB8), SDHB and cytochrome c oxidase 1 (MTCO1). Thus, SPARC plays a crucial role in the proliferation and differentiation of C2C12 and may be involved in the link between the ECM remodeling and mitochondrial function.
Proper citation: Aicha Melouane, Mayumi Yoshioka, Jonny St-Amand 2017. Implication of SPARC in the modulation of the extracellular matrix and mitochondrial function in muscle cells. protocols.io dx.doi.org/10.17504/protocols.io.jsycnfw Copy
Authors: Jarmo Koskiniemi, Marja-Liisa Koljonen, Tuomas Leinonen
Summary: In this protocol we describe laboratory methods for DNA extraction and multiplex genotyping of Atlantic salmon with microsatellite markers. The protocol has been used in several studies at the University of Helsinki and the Natural Resources Institute Finland (Luke). Publications from these studies are listed in the attachment.
Proper citation: Jarmo Koskiniemi, Marja-Liisa Koljonen, Tuomas Leinonen 2020. University of Helsinki and Natural Resources Institute Finland (Luke) protocol for DNA extraction and multiplex PCR genotyping of 17 microsatellites for Atlantic salmon (Salmo salar L.).. protocols.io dx.doi.org/10.17504/protocols.io.bp7umrnw Copy
Authors: Stephanie Cullum
Group: Plantae
Summary: In order to take chlorophyll fluroscence images of cowpea accessions, leaf disks need to be collected and placed randomly onto a leaf disk tray. This protocol explains how.
Proper citation: Stephanie Cullum 2018. Cowpea Leaf disk collection. protocols.io dx.doi.org/10.17504/protocols.io.rspd6dn Copy
Authors: Peng Wang, Hongjian Ni, Ruihe Wang
Proper citation: Peng Wang, Hongjian Ni, Ruihe Wang 2018. Fig 8. Effect of pressure pulse amplitude on WOB at different frequencies. A. f =2 Hz. B. f =6 Hz. C. f =10 Hz. protocols.io dx.doi.org/10.17504/protocols.io.ke9cth6 Copy
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