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Authors: Sebastian Kirchner, Robert Rauscher, Andreas Czech, Zoya Ignatova
Group: Ignatova Lab
Summary: This protocol describes the transfection of mammalian cells with in vitro transcribed, unmodified, non-charged tRNAs. We have used this method successfully to raise intrinsic tRNA concentrations in HeLa1 and N2a cells2. Furthermore, tRNAs synthesised as described in this protocol have been shown to be translation competent1.References1. Kirchner et al. Alteration of Protein Function by a Silent Polymorphism Linked to tRNA Abundance. PLoS Biology. 2017, in press.2. Girstmair et al. Depletion of Cognate Charged Transfer RNA Causes Translational Frameshifting within the Expanded CAG Stretch in Huntingtin. Cell Reports. 2013, 3(1):148-59. DOI: http://dx.doi.org/10.1016/j.celrep.2012.12.019
Proper citation: Sebastian Kirchner, Robert Rauscher, Andreas Czech, Zoya Ignatova 2017. Upregulating tRNAs in Mammalian Cells through Transfection of In Vitro Transcribed tRNAs. protocols.io dx.doi.org/10.17504/protocols.io.hetb3en Copy
Authors: David Eccles
Summary: This protocol is for preparing long reads for stranded mapping, as an intermediate step for additional protocols:Aligning strand-oriented sequences to a transcriptome for transcript / gene countingAligning strand-oriented sequences to a genome for confirmatory QCInput(s): demultiplexed fastq files (see protocol Demultiplexing Nanopore reads with LAST), adapter file (containing strand-sensitive adapter sequences)Output(s): oriented read files, as gzipped fastq files
Proper citation: David Eccles 2019. Preparing Reads for Stranded Mapping. protocols.io dx.doi.org/10.17504/protocols.io.43qgymw Copy
Authors: Magdalena Gutowska, Sebastian Sudek, Alexandra Worden, Brateen Shome, Tadhg Begley
Group: Worden Lab
Summary: We use the described methods for determining the internal cellular quota of thiamin and its
different phosphorylation states in marine algae, in the below case for the haptophyte Emiliania
huxleyi. The chemical assay used – the thiochrome assay - was developed in the 1930s but has
been modified over the decades to include instrumentation with improved detection (Backstrom et
al. 1995, Reddick et al. 2001). The assay is based on cyclization/oxidation of thiamin to
thiochrome under basic conditions in the presence of an oxidizing agent such as potassium
ferricyanide. Thiochrome is intensely fluorescent and easy to detect and quantify with high
sensitivity in an HPLC assay. This method has previously been used to determine cellular quotas
of thiamin and its different phosphorylation states in algae from brackish waters (Pinto et al. 2002,
Sylvander et al. 2013).
Proper citation: Magdalena Gutowska, Sebastian Sudek, Alexandra Worden, Brateen Shome, Tadhg Begley 2017. Quantifying Thiamin Cellular Quotas in Algae. protocols.io dx.doi.org/10.17504/protocols.io.j6ncrde Copy
Authors: Bryon Drown
Group: Kelleher Research Group
Summary: Tris buffered saline (TBS) is a commonly used buffer with a wide range of applications in biochemistry. It is often more convenient to prepare a 10X solution so that additional components can be added when preparing the buffer on demand.
Proper citation: Bryon Drown 2020. 10X TBS Buffer. protocols.io dx.doi.org/10.17504/protocols.io.bd85i9y6 Copy
Authors: Caio Maximino
Group: Fish behavior and physiology, Medicinal Plants Southeastern Pará Research Group
Summary: The light/dark test is increasingly being adopted as one of the main behavioral assays in zebrafish neuroscience research. It is based on the innate preference of adult zebrafish for a black vs. a white compartment. Modifications and extensions of the protocol increased its breadth, but also its heterogeneity. The protocol presented here involves confining the animal in a central compartment for a initial 3-min acclimation period, followed by free exploration of an acryllic tank with a black and a white compartment. In addition to observing the spatial distribution of the animal, "ethological" variables (risk assessment, erratic swimming, freezing, thigmotaxis) are also manually scored in the white conpartment; these endpoints are selectively sensitive to drug treatments. The protocol is intended to measure anxiety-like behavior, with anxiogenic effects decreasing time on white and increasing risk assessment and thigmotaxis; some treatments also increase erratic swimming and freezing.
Proper citation: Caio Maximino 2018. Light/dark preference test for adult zebrafish (Danio rerio). protocols.io dx.doi.org/10.17504/protocols.io.srfed3n Copy
Authors: Aurore Mathys
Proper citation: Aurore Mathys 2019. Spectral photogrammetry protocol. protocols.io dx.doi.org/10.17504/protocols.io.v52e88e Copy
Authors: Gabriel Magalhães Nunes Guimarães
Summary: A single prospective cohort of women submitted to cesarean section who received spinal anesthesia will be assessed for the proposed risk factors and the incidence of nausea or vomiting will be observed during the first 48 hours.
Proper citation: Gabriel Magalhães Nunes Guimarães 2018. NCT03171688 - clinical protocol (PT-BR). protocols.io dx.doi.org/10.17504/protocols.io.q9mdz46 Copy
Authors: Zymo Research
Group: Zymo Research - The Epigenetics Company
Summary: The Zyppy™ Plasmid Miniprep Kit features a Pellet-Free™ modified alkaline lysis method that bypasses bacterial culture centrifugation and resuspension steps common to classical plasmid preparation procedures. Simply add the uniquely formulated 7X Lysis Buffer directly to your bacterial culture, neutralize, then purify using our Fast-Spin column technology (alternatively, the samples may be processed by the classical centrifugation method). Additionally, the innovative colored buffers included in the kit permit error-free visualization identification of complete bacterial cell lysis and neutralization.
Proper citation: Zymo Research 2016. Zyppy™ Plasmid Miniprep Kit. protocols.io dx.doi.org/10.17504/protocols.io.gi3bugn Copy
Authors: Benjamin Lau, Abrizah Othman
Summary: Protein solubility is a critical prerequisite to any proteomics analysis. Combination of urea/thiourea and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) have been routinely used to enhance protein solubilization for oil palm proteomics studies in recent years. The goals of these proteomics analysis are essentially to complement the knowledge regarding the regulation networks and mechanisms of the oil palm fatty acid biosynthesis. Through omics integration, the information is able to build a regulatory model to support efforts in improving the economic value and sustainability of palm oil in the global oil and vegetable market. Our study evaluated the utilization of sodium deoxycholate as an alternative solubilization buffer/additive to urea/thiourea and CHAPS. Efficiency of urea/thiourea/CHAPS, urea/CHAPS, urea/sodium deoxycholate and sodium deoxycholate buffers in solubilizing the oil palm (Elaeis guineensis var. Tenera) mesocarp proteins were compared. Based on the protein yields and electrophoretic profile, combination of urea/thiourea/CHAPS were shown to remain a better solubilization buffer and additive, but the differences with sodium deoxycholate buffer was not significant. Furthermore, a deeper mass spectrometric and statistical analyses on the identified proteins and peptides from all the evaluated solubilization buffers revealed that sodium deoxycholate had similar efficiency in solubilizing proteins from oil palm mesocarps. However, it was worth to note that observed limitations to the application of sodium deoxycholate in protein solubilization were the interference with protein quantitation and only 70% of the total identified proteins were shared between the buffers. The former limitation could be rectified through a 4-fold dilution but further works are needed to determine the importance of the remaining 30% unique proteins. All the proteomics data are available via ProteomeXchange with identifier PXD013255. In conclusion, sodium deoxycholate is applicable in the solubilization of proteins extracted from oil palm mesocarps to an efficiency level comparable to that of urea/thiourea/CHAPS buffer.
Proper citation: Benjamin Lau, Abrizah Othman 2019. Evaluation of sodium deoxycholate as solubilization buffer for oil palm proteomics analysis. protocols.io dx.doi.org/10.17504/protocols.io.434gyqw Copy
Authors: Alexander H Wilcox, Erik Delwart, Samuel L Díaz Muñoz
Group: Diaz-Munoz Lab
Proper citation: Alexander H Wilcox, Erik Delwart, Samuel L Díaz Muñoz 2019. Preparation of dsRNA viruses for next-generation sequencing. protocols.io dx.doi.org/10.17504/protocols.io.ugnetve Copy
Authors: Alan Daugherty, Jing Liu, Hong Lu
Group: Sangderk Lee lab
Summary: Suprarenal region of the abdominal aorta is the portion from the last intercostal artery to the right renal artery.In the Angiotensin II infused mouse model, abdominal aortic aneurysms are located in this region. Maximal outer width of the suprarenal aorta is a major parameter to define abdominal aortic aneurysms. Fifty % or more increase of this parameter, compared to control (saline infusion), is defined as an abdominal aortic aneurysm.
Proper citation: Alan Daugherty, Jing Liu, Hong Lu 2017. In Situ Quantification of Abdominal Aortic Aneurysms. protocols.io dx.doi.org/10.17504/protocols.io.ix7cfrn Copy
Authors: Judy Northill, Ian Mackay
Group: Public Health Virology, Forensic and Scientific Services, Coronavirus Method Development Community
Summary: A nested RT-PCR targeting the RdRp region of the sub-genus Sarbecovirus. The primers are modified from the pan-coronavirus RT-PCR published by Hu et al. 2017 to be more specific to SARS-CoV-2.Assay may be used in resource poor settings where real-time cyclers are not available.Sanger sequencing can be used to confirm SARS-CoV-2 where WGS is not available or where WGS fails due to poor quality sample.
Proper citation: Judy Northill, Ian Mackay 2020. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RdRp nested RT-PCR. protocols.io dx.doi.org/10.17504/protocols.io.bdcpi2vn Copy
Authors: Doha Mohammed Afifi, Amal Hassan Abdel-Rahman, Safa Fathy Abd El-Ghany
Proper citation: Doha Mohammed Afifi, Amal Hassan Abdel-Rahman, Safa Fathy Abd El-Ghany 2018. Apoptotic Effect of Crude Scorpion Venom on Head and Neck Squamous Cell Carcinoma Cell Line versus Normal Human Epithelial Cell Line (In-vitro Study). protocols.io dx.doi.org/10.17504/protocols.io.t5aeq2e Copy
Authors: Ibrahim Ilik, Tugce Aktas, Daniel Maticzka, Rolf Backofen, Asifa Akhtar
Summary: Determination of the in vivo binding sites of RNA-binding proteins (RBPs) is paramount to understanding their function and how they affect different aspects of gene regulation. With hundreds of RNA-binding proteins identified in human cells, a flexible, high-resolution, high-throughput, highly multiplexible and radioactivity-free method to determine their binding site has not been described to date. Here we report FLASH (Fast Ligation of RNA after some sort of Affinity Purification for High-throughput Sequencing), which uses a special adapter design and an optimized protocol to determine protein-RNA interactions in living cells. The entire FLASH protocol, starting from cells-on-plates to a sequencing library, takes 1.5 days. We demonstrate the flexibility, speed and versatility of FLASH by using it to determine RNA targets of both tagged and endogenously expressed proteins under diverse conditions in vivo.
Proper citation: Ibrahim Ilik, Tugce Aktas, Daniel Maticzka, Rolf Backofen, Asifa Akhtar 2019. FLASH. protocols.io dx.doi.org/10.17504/protocols.io.zv9f696 Copy
Authors: Manuel Liebeke, Janine Beckmann
Group: Mass Spectrometry at MPI-Bremen, Metabolomics Protocols & Workflows
Summary: Depending on tissue and analytes that will be analysed different matrices can be used. The following 2 matrices are first steps to analyse lipids (SDHB) and peptides (CHCA). Matrix concentration and solvent ratio as well as instrument settings can be adjusted specifically to tissue and analytes.
Proper citation: Manuel Liebeke, Janine Beckmann 2018. Matrix Deposition using the SunChrom Sprayer. protocols.io dx.doi.org/10.17504/protocols.io.ug5ety6 Copy
Authors: Yoshihisa Hirakawa
Group: Protist Research to Optimize Tools in Genetics (PROT-G)
Proper citation: Yoshihisa Hirakawa 2016. Transformtion of Perkinsus marinus by Amaxa and Bio-Rad. protocols.io dx.doi.org/10.17504/protocols.io.e5nbg5e Copy
Authors: Celeste Karch, Rita Martinez, Jacob Marsh
Group: Neurodegeneration Method Development Community
Proper citation: Celeste Karch, Rita Martinez, Jacob Marsh 2019. Thawing iPSC Plate. protocols.io dx.doi.org/10.17504/protocols.io.x8ufrww Copy
Authors: Ken Youens-Clark, Bonnie Hurwitz
Group: Hurwitz Lab, Metafunc Course 2017
Summary: Set up ssh keys to make login to the HPC easy!
Proper citation: Ken Youens-Clark, Bonnie Hurwitz 2017. Github ssh keys. protocols.io dx.doi.org/10.17504/protocols.io.jh7cj9n Copy
Authors: Shaina Robbins, Sean Nieves
Group: SPARC
Proper citation: Shaina Robbins, Sean Nieves 2020. 0.1% Cresyl Violet Stain (pH 4.3). protocols.io dx.doi.org/10.17504/protocols.io.wcjfaun Copy
Authors: Kat Dawson
Group: Orphan Lab
Proper citation: Kat Dawson 2017. PFA fixation and Percoll prep of sediments. protocols.io dx.doi.org/10.17504/protocols.io.g5nby5e Copy
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