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Authors: Amy Zimmerman
Group: VERVE Net, Worden Lab
Summary: Purpose: To evaluate the influence of nutrient availability on cellular elemental composition (quotas and stoichiometry) in marine picoeukaryotes.Elemental quotas and ratios are assessed under nutrient replete and deplete conditions at the same time to minimize potential variation between experiments. Cells from an exponentially growing culture are concentrated by centrifugation, washed and re-suspended in a small volume of nutrient deplete media. These concentrated cells are used to inoculate triplicate replete and deplete culture flasks at a starting density corresponding to early-exponential growth. Culture growth is monitored daily. When the mean growth rate of the nutrient deplete treatment is half or less of the replete treatment (GRDEP/GRREP
Proper citation: Amy Zimmerman 2016. Nutrient deplete/replete algal culture for elemental analysis. protocols.io dx.doi.org/10.17504/protocols.io.fcdbis6 Copy
Authors: Israel Aguilar Ordoñez
Group: Whole genome variation in 27 Mexican indigenous populations, demographic and biomedical insights
Summary: 'nf-vcf-cataloguer' is a tool, implemented in Nextflow, that generates a general table description in TSV format of the description of each category and subgroup of a VCF with the extended annotation made by VEP. Furthermore, it plots each subset of the consequences of variants.
Proper citation: Israel Aguilar Ordoñez 2020. nf-vcf-cataloguer. protocols.io dx.doi.org/10.17504/protocols.io.bkmzku76 Copy
Authors: Ye Zheng, Kami Ahmad, Steven Henikoff
Summary: This tutorial is designed for processing and analyzing CUT&Tag data following the Benchtop CUT&Tag V.3 protocol. The illustration data used in this tutorial is the profiling of histone modifications in the human lymphoma K562 cell line, but the tutorial is generally applicable to any chromatin protein, including transcription factors, RNA polymerase II, and epitope-tagged proteins. For reproducible analysis, this tutorial is also available on GitHub at https://yezhengstat.github.io/CUTTag_tutorial/.
Proper citation: Ye Zheng, Kami Ahmad, Steven Henikoff 2020. CUT&Tag Data Processing and Analysis Tutorial. protocols.io dx.doi.org/10.17504/protocols.io.bjk2kkye Copy
Authors: Randie Bundy
Summary: This is a protocol for making a trace metal clean 0.5 mol L-1 EPPS (N-(2-hydroxyethyl)piperazine-N-(3-propanesulfonic acid) solution for dissolved cobalt analyses.
Proper citation: Randie Bundy 2016. EPPS recipe for dissolved cobalt analyses. protocols.io dx.doi.org/10.17504/protocols.io.evcbe2w Copy
Authors: Yuan Yao, morigen
Proper citation: Yuan Yao, morigen 2018. Flow cytometry. protocols.io dx.doi.org/10.17504/protocols.io.p9ndr5e Copy
Authors: Binnypreet Kaur1, 2, Drahomíra Faktorová1, 2, , Priscila Peña-Diaz1 and Julius Lukeš1, 2
Group: Julius Lukes
Summary: E-mail: [email protected]
Proper citation: Binnypreet Kaur1, 2, Drahomíra Faktorová1, 2, , Priscila Peña-Diaz1 and Julius Lukeš1, 2 2018. Diplonema Genomic DNA isolation. protocols.io dx.doi.org/10.17504/protocols.io.hfyb3pw Copy
Authors: Donald McClain
Group: Diabetic Complications Consortium
Summary: This is the standard protocol for measuring insulin action in terms of total body glucose disposal. It is the "gold standard" for quantifying insulin resistance/sensitivity.Diabetic Complications:
Proper citation: Donald McClain 2019. Glucose "Clamp" for measurement of whole body insulin-stimulated glucose disposal. protocols.io dx.doi.org/10.17504/protocols.io.7s9hnh6 Copy
Authors: Michael Bokoch, Claus Niemann, Dieter Adelmann, Rishi Kothari
Group: UCSF Transplant Anesthesia Research Group (TARG)
Summary: Acute kidney injury (AKI), or worsening kidney function, is a common complication after liver transplantation (20-90% in published studies). Patients who experience AKI after liver transplantation have higher mortality, increased graft loss, longer hospital and intensive care unit stays, and more progression to chronic kidney disease compared with those who do not. In this study, half of the participants will have their body temperature cooled to slightly lower than normal (mild hypothermia) for a portion of the liver transplant operation, while the other half will have their body temperature maintained at normal. The study will evaluate if mild hypothermia protects from AKI during liver transplantation.This study is a single-blinded, randomized controlled trial of mild hypothermia during liver transplantation to provide protection from AKI. Participants will be randomized to normothermia (36.5-37.5 °C) versus mild hypothermia (34-35 °C) during a portion of the liver transplant operation. The protocol is based on preliminary data from rodent models showing that hypothermia protects the kidneys from ischemia-reperfusion injury, as well as studies in deceased organ donors showing that cooling improves post-transplant organ function. Temperature will be maintained with standard techniques plus a minimally-invasive esophageal cooling device that is approved by the U.S. Food and Drug Administration. The investigators hypothesize that mild hypothermia will reduce the incidence and severity of AKI after LTx. Standard surrogates (e.g., change in serum creatinine, need for initiation of dialysis) and biomarkers will be used to assess the severity of kidney injury.
Proper citation: Michael Bokoch, Claus Niemann, Dieter Adelmann, Rishi Kothari 2020. Mild Hypothermia to prevent Acute kidney injury in Liver Transplantation (MHALT) Trial - Statistical Analysis Plan for the Interim Analysis, v1.0 . protocols.io dx.doi.org/10.17504/protocols.io.bf5kjq4w Copy
Authors: Allen Institute for Brain Science
Group: BICCN, Allen Institute for Brain Science
Summary: This protocol describes the preparation of 1x3 glass slides for mounting embedded mouse brain samples for subsequent imaging and sectioning on TissueCyte 1000. 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 2020. Slide Preparation for Mounting Samples for TissueCyte 1000 Sectioning and Imaging. protocols.io dx.doi.org/10.17504/protocols.io.bdvni65e Copy
Authors: Anna Behle
Group: Axmann Lab
Summary: Protocol for radioactive labelling of RNA using T7-Polymerase and α-[32P]-UTP.
Proper citation: Anna Behle 2017. Radioactive in vitro transcription. protocols.io dx.doi.org/10.17504/protocols.io.gudbws6 Copy
Authors: Bulent Arman Aksoy, Pinar Aksoy, Jeff Hammerbacher
Group: Hammer Lab
Proper citation: Bulent Arman Aksoy, Pinar Aksoy, Jeff Hammerbacher 2018. Preparing primary T cells for fluorescence microscopy. protocols.io dx.doi.org/10.17504/protocols.io.vede3a6 Copy
Authors: LGC Biosearch Technologies
Summary: Stellaris RNA FISH protocol to simultaneously label adherent cells with IF and RNA FISH.
Proper citation: LGC Biosearch Technologies 2016. Stellaris® RNA FISH Simultaneous IF + FISH in Adherent Cells Protocol. protocols.io dx.doi.org/10.17504/protocols.io.ek8bczw Copy
Authors: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani , Ana Herrmann, Angelo Piato
Group: Fish behavior and physiology
Summary: Zebrafish are incresingly used as a model animal in neuroscience research. Here we describe a protocol to quantify the total amount of proteins in zebrafish brain tissue.
Proper citation: Adrieli Sachett, Matheus Gallas-Lopes, Greicy M M Conterato, Radharani , Ana Herrmann, Angelo Piato 2020. Protein quantification protocol optimized for zebrafish brain tissue (Bradford method). protocols.io dx.doi.org/10.17504/protocols.io.bjnfkmbn Copy
Authors: Rebecca Maher
Proper citation: Rebecca Maher 2018. Instructions for Planar Area Analysis using CPCe 4.1 Software. protocols.io dx.doi.org/10.17504/protocols.io.pmvdk66 Copy
Authors: Dr. Steven Wilhelm
Group: The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs), CyanoHABs
Summary: Please contact Dr. Steven Wilhelm ([email protected]) for additional information regarding this protocol.Modified from Bold 1949, Bischoff and Bold 1963
Proper citation: Dr. Steven Wilhelm 2017. BBM Media. protocols.io dx.doi.org/10.17504/protocols.io.h97b99n Copy
Authors: Michiyo Tsuru
Group: Clinical Proteomics and Gene Therapy Laboratory
Summary: We generated a miR-340 probe by tagging Has-miR-340 aatcaG(L)t5(L)aT(L)tG(L)cT(L)ttataa_N(6)_Y with Alexa Fluor 488. LNA-in situ hybridization (ISH) was performed according to the manufacturer’s instructions (http://www.exiqon.com/mirna-ish-kit). Sections of FFPE tissues of human OPLL and null mice (4-µm-thick) were fixed with 4 % PFA in PBS for 20 min at room temperature, washed with PBS (3 × 5 min), treated with 0.5 % Triton X-100 (10 min at 4 °С), and briefly washed with PBS followed by two washes (10 min each) in saline-sodium citrate buffer (2× SSC, 0.3 M NaCl, 0.03 M Na3С6Н5О7, рН 7.0). The sections were then digested with 15 μg/mL proteinase K (Exiqon, Vedbaek, Denmark) at 37 °C for 10 min and rinsed for 3 × 5 min in PBS. Hybridization was performed in a humid chamber for 18 h at 65 °C. In situ hybridization of miR-340 was conducted using a miRCURY LNA microRNA ISH Optimization kit (Exiqon, Vedbaek, Denmark). miR-340-Alexa Fluor 488 and bone morphogenetic protein 2 (Alexa Fluor 555) were detected in human OPLL tissue by in situ hybridization and immunocytochemistry, respectively. Nuclei were counterstained with DAPI. After staining, the tissues were observed by fluorescence microscopy (BZ-X700, Keyence, Osaka, Japan).
Proper citation: Michiyo Tsuru 2018. In situ hybridization. protocols.io dx.doi.org/10.17504/protocols.io.j9hcr36 Copy
Authors: Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger
Group: High molecular weight DNA extraction from all kingdoms
Summary: DNA extractions often contain impurities which limit the output of long-read sequencing technologies. Here a protocol is provided which removes impurities and size selects for longer fragments. To remove residual RNA and protein, an additional RNAse A and Proteinase K treatment is performed. A clean-up with chloroform: isoamyl alcohol (24:1) removes these proteins and other hydrophobic organics such as lipids. A low volume ethanol precipitation and wash is used to concentrate the DNA, hopefully also reducing polysaccharides. Finally, a Short Read Eliminator (SRE) kit by Circulomics is utilised for size selection, which also appears to clean the DNA. This has been trialled for the sorghum rot fungus Macrophomina phaseolina, providing highly promising results with an Oxford Nanopore MinION. One strain yielded 13.71 Gbases with an N50 of 21.75 kb, another strain yielded 9.72 Gbases with an N50 of 43.50 kb. Similar results are expected across many organisms, but have not been tested.
Proper citation: Ashley Jones, Neeraj Purushotham, Jamila Nasim, Benjamin Schwessinger 2019. DNA clean-up and size selection for long-read sequencing. protocols.io dx.doi.org/10.17504/protocols.io.6kahcse Copy
Authors: Cristina Rodrigues Gabriel Sales, Anabela Silva, Elizabete Carmo-Silva
Summary: This protocol uses five reactions to couple RuBP carboxylation and 3-PGA formation to NADH oxidation to measure Rubisco activity, based on Scales et al. (2014).
Proper citation: Cristina Rodrigues Gabriel Sales, Anabela Silva, Elizabete Carmo-Silva 2020. NADH-linked microtiter plate-based assay for measuring Rubisco activity & activation state – PK-LDH. protocols.io dx.doi.org/10.17504/protocols.io.bf9rjr56 Copy
Authors: Bieke Tack
Summary: Standard operating procedures for Salmonella blood culture surveillance of the unit of tropical bacteriology at the Institute of Tropical Medicine in Antwerp (Belgium):- Blood culture work-up- Pathogen identification- Salmonella serotyping- Antibiotic susceptibility testing
Proper citation: Bieke Tack 2019. Salmonella blood culture surveillance: SOP's ITM. protocols.io dx.doi.org/10.17504/protocols.io.9rah52e Copy
Authors: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward
Group: Neurodegeneration Method Development Community
Summary: Following Day 3 replating, i3Neurons should be cultured for at least 1 week before lysis, fixation, or other experimental endpoint, and they remain viable for at least 1 month with proper maintenance. This protocol covers medium conditions, coating of tissue culture dishes with synthetic polymers, recommended plating densities, and maintenance procedures for long-term culture and for specific experimental applications.Cortical Neuron Culture Medium (CM) is sufficient to promote the maturation and longterm maintenance of i3Neurons in culture. i3Neurons express general markers of cortical neurons, as well as specific pre- and postsynaptic markers of glutamatergic excitatory cortical neurons. Since these cells are post-mitotic after 3 days of differentiation (see Basic Protocol 5) and prefer neuron-conditioned medium to fresh medium, maintenance conditions for these cultures are minimal. Generally, half-medium changes every 7 days with fresh, pre-warmed CM are sufficient for culturing beyond d10 (7 days after replating). Neuron attachment and growth also require a strongly adhesive substrate. Coating plates with synthetic polymers such as poly-L-ornithine (PLO) is necessary for i3Neuron attachment, viability, and successful outgrowth.
Proper citation: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward 2019. CULTURING i3NEURONS (Basic Protocol 6). protocols.io dx.doi.org/10.17504/protocols.io.5jpg4mn Copy
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