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Authors: Elisa Granato
Group: Elisa Granato
Proper citation: Elisa Granato 2020. Quick'n'Dirty electrocompetent E. coli cells . protocols.io dx.doi.org/10.17504/protocols.io.bjpykmpw Copy
Authors: Tanja Bosak
Group: Bosak Lab
Proper citation: Tanja Bosak 2020. BG11 hypersaline medium. protocols.io dx.doi.org/10.17504/protocols.io.bkcmksu6 Copy
Authors: Evripidis Gkanias, Benjamin Risse, Michael Mangan, Barbara Webb
Summary: InsectVisionComputer Vision Toolbox for Insect VisionVersion 1.0.1This version has been created as support for the article:Gkanias E., Risse B., Mangan M., and Webb B. (2019) From skylight input to behavioural output: a computational model of the insect polarised light compass. PLOS Computational Biology.To be able to run all the experiments and replicate the results presented in the article, the compmodels package is needed. If you don't have access to this package, please contact the authors.Clone both repositories and set the compmodels as a dependence to the insectvision package.To see the results, it is not necessary to run the code. By simply opening the notebooks/plos.ipynb file, the plots should be automatically generated for you to observe them.To create the plots by yourself, you need to start a Jupyter notebook kernel at the root of the package. Then run the notebooks/plos.ipynb file, which already contains all the plots and the respective code to replicate them. Some plots (especially the ones related to the global optimisation) may need a long time to run; this does not mean that they do not work.All the code has been implemented by Evripidis Gkanias.You can go the github repository by following this link. The release version is attached and it can also be found here.Copyright © 2019 Gkanias el al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Proper citation: Evripidis Gkanias, Benjamin Risse, Michael Mangan, Barbara Webb 2019. Insect Vision 1.0.1. protocols.io dx.doi.org/10.17504/protocols.io.y9hfz36 Copy
Authors: Colin Daniel
Proper citation: Colin Daniel 2020. Ab Lysis Buffer . protocols.io dx.doi.org/10.17504/protocols.io.bft7jnrn Copy
Authors: Ashley Humphrey
Group: Protist Research to Optimize Tools in Genetics (PROT-G), The Aquatic Microbial Ecology Research Group - AMERG (The Buchan, Zinser and Wilhelm labs)
Summary: Stock vitamin solution for ESAW Media for Marine Phytoplankton
Proper citation: Ashley Humphrey 2016. Vitamin Solution for ESAW Media for Marine Phytoplankton. protocols.io dx.doi.org/10.17504/protocols.io.ge9bth6 Copy
Authors: LI-COR Biosciences
Group: LI-COR Biosciences
Summary: The Odyssey Fc Imager, with 600 channel capabilities, can image agarose gels stained with popular DNA stains, such as ethidium bromide and SYBR Safe DNA stain, with sub-nanogram sensitivity. The Odyssey Fc Imager contains a 532 nm diffuse source with an excitation maximum of 520 nm and a detection maximum of 600 nm. These instrument parameters are within the range of the excitation and emission wavelengths of ethidium bromide (Ex/Em = 302 & 518/605 nm1,2) and other visible fluorescent nucleic acid stains and provide a sensitive gel documentation option.SYBR Safe DNA stain (Ex/Em = 502/530 nm) has also been tested on the Odyssey Fc Imager (using the 600 channel) with sensitivities exceeding ethidium bromide detection. The maximum fluorescence emission wavelength of SYBR Safe is very close to the maximum excitation wavelength. However, the Odyssey Fc 600 channel collects excitation light at a wavelength 50 nm higher than the maximum excitation wavelength of SYBR Safe. These instrument properties decrease the background and improve the signal-to-noise ratio for nucleic acid detection.Specific instructions are given in this technical note for ethidium bromide and SYBR Safe use. There are a variety of commercial DNA stains that may be appropriate for fluorescent imaging with the Odyssey Fc 600 channel. SYBR Green I (Life Technologies), GelStar (FMC), Gel Red™ (Biotium), Gel Green™ (Biotium) and Nancy-520 (Sigma) stains have also been tested at LI-COR (see example images on page 7). Other nucleic acid binding stains may also be compatible with the Odyssey Fc Imager. Please check the excitation and emission spectra of each stain.Developed for: Odyssey Fc Imaging System
Proper citation: LI-COR Biosciences 2018. DNA Separation and Detection on Agarose Gels/Post-Electrophoresis Staining Protocol. protocols.io dx.doi.org/10.17504/protocols.io.q92dz8e Copy
Authors: Lenny Teytelman, Anjuli Manche, Alexei Stoliartchouk
Group: protocols.io news
Proper citation: Lenny Teytelman, Anjuli Manche, Alexei Stoliartchouk 2016. How to create a new protocol. protocols.io dx.doi.org/10.17504/protocols.io.evxbe7n Copy
Authors: Vinay Saunders, David I Wilson
Summary: Procotol describing RNA extraction from dissected human tissue followed by reverse transcription to cDNA, PCR for detection of TMSB4X and separation of PCR products by agarose gel.
Proper citation: Vinay Saunders, David I Wilson 2018. RNA extraction, reverse transcription and PCR for TMSB4X. protocols.io dx.doi.org/10.17504/protocols.io.rvbd62n Copy
Authors: Luke Schwerdtfeger, Elizabeth Ryan, Stuart Tobet
Summary: Organotypic tissue slices provide seminatural, three-dimensional microenvironments for use in ex vivo study of specific organs and have advanced investigative capabilities compared with isolated cell cultures. Several characteristics of the gastrointestinal tract have made in vitro models for studying the intestine challenging, such as maintaining the intricate structure of microvilli, the intrinsic enteric nervous system, Peyer’s patches, the microbiome, and the active contraction of gut muscles. In the present study, an organotypic intestinal slice model was developed that allows for functional investigation across regions of the intestine. Intestinal tissue slices were maintained ex vivo for several days in a physiolog- ically relevant environment that preserved normal enterocyte struc- ture, intact and proliferating crypt cells, submucosal organization, and muscle wall composure. Cell death was measured by a membrane- impermeable DNA binding indicator, ethidium homodimer, and less than 5% of cells were labeled in all regions of the villi and crypt epithelia at 24 h ex vivo. This tissue slice model demonstrated intact myenteric and submucosal neuronal plexuses and functional intersti- tial cells of Cajal to the extent that nonstimulated, segmental contrac- tions occurred for up to 48 h ex vivo. To detect changes in physio- logical responses, slices were also assessed for segmental contractions in the presence and absence of antibiotic treatment, which resulted in slices with lesser or greater amounts of commensal bacteria, respec- tively. Segmental contractions were significantly greater in slices without antibiotics and increased native microbiota. This model ren- ders mechanisms of neuroimmune-microbiome interactions in a com- plex gut environment available to direct observation and controlled perturbation.
Proper citation: Luke Schwerdtfeger, Elizabeth Ryan, Stuart Tobet 2019. An organotypic slice model for ex vivo study of neural, immune, and microbial interactions of mouse intestine. protocols.io dx.doi.org/10.17504/protocols.io.xjvfkn6 Copy
Authors: Katharine H. D. Crawford, Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Jesse D. Bloom
Group: Coronavirus Method Development Community
Summary: SARS-CoV-2 enters cells using its Spike protein, which is also the main target of neutralizing antibodies. Therefore, assays to measure how antibodies and sera affect Spike-mediated viral infection are important for studying immunity. Because SARS-CoV-2 is a biosafety-level-3 virus, one way to simplify such assays is to pseudotype biosafety-level-2 viral particles with Spike. Such pseudotyping has now been described for single-cycle lentiviral, retroviral and VSV particles, but the reagents and protocols are not widely available. Here we detail how to effectively pseudotype lentiviral particles with SARS-CoV-2 Spike and infect 293T cells engineered to express the SARS-CoV-2 receptor, ACE2. We also make all the key experimental reagents available in the BEI Resources repository of ATCC and the NIH. Furthermore, we demonstrate how these pseudotyped lentiviral particles can be used to measure the neutralizing activity of human sera or plasma against SARS-CoV-2 in convenient luciferase-based assays, thereby providing a valuable complement to ELISA-based methods that measure antibody binding rather than neutralization.
Proper citation: Katharine H. D. Crawford, Rachel Eguia, Adam S. Dingens, Andrea N. Loes, Jesse D. Bloom 2020. Pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays. protocols.io dx.doi.org/10.17504/protocols.io.bfghjjt6 Copy
Authors: Ziqiang Chen
Group: GigaScience Press, BGI
Summary: BGISEQ-500 is a desktop sequencer developed by BGI. This protocol adjusts the process in order to apply the 10X contruction to the BGISEQ-500 rather than illumina.
Proper citation: Ziqiang Chen 2019. BGISEQ-500 10X library construction. protocols.io dx.doi.org/10.17504/protocols.io.3jagkie Copy
Authors: Kristen Upton, Robyn T. Sussman, Khushbu Patel, Gregory P. Way, Rebecca N. Adams, Gregory I. Sacks, Rebecca N. Adams, Paolo Fortina, John M. Maris, Jo Lynne Rokita
Summary: This protocol explains the process of how we collected MYCN, MYC, and Histone ChIP-Seq data, as well as ATAC-Seq data for neuroblastoma cell lines. This protocol is comprised of three sections: Cell Growth and Expansion for care of neuroblastoma cell lines, ChIP-Seq protocol, and ATAC-Seq Protocol. Table 1, within the document, outlines which data was collected for each cell line.
Proper citation: Kristen Upton, Robyn T. Sussman, Khushbu Patel, Gregory P. Way, Rebecca N. Adams, Gregory I. Sacks, Rebecca N. Adams, Paolo Fortina, John M. Maris, Jo Lynne Rokita 2019. Epigenomic profiling of neuroblastoma cell lines. protocols.io dx.doi.org/10.17504/protocols.io.6r3hd8n Copy
Authors: Josh Quick, Kirstyn Brunker
Summary: This is a ‘one-pot ligation’ protocol for Oxford Nanopore native barcoded ligation libraries using shearing.
Proper citation: Josh Quick, Kirstyn Brunker 2020. One-pot native barcoding of amplicons (Ultra II AMII ligation). protocols.io dx.doi.org/10.17504/protocols.io.bdaqi2dw Copy
Authors: Brent A. Biddy, Wenjun Kong, Kenji Kamimoto, Chuner Guo, Sarah Waye, Tao Sun, Samantha Morris
Group: Human Cell Atlas Method Development Community, Morris Lab
Summary: Single-cell technologies are offering unprecedented insight into complex biology, revealing the behavior of rare cell populations that are typically masked in bulk population analyses. One current limitation of single-cell approaches is that lineage relationships are lost as a result of cell processing, restricting interpretations of the data collected. Elegant computational approaches have been developed in an effort to infer these missing observations, but it remains a challenge to reconstruct true reprogramming trajectories using these tools. Although sophisticated lineage tracing solutions to connect cell history with fate are emerging, these protocols are either not compatible with high-throughput scRNA-seq, or require genome editing strategies that are not readily deployed in some systems. Here, our protocol describes a single-cell resolution clonal tracking approach, ‘CellTagging’, based on combinatorial cell indexing, permitting the parallel capture of lineage information and cell identity. CellTagging integrates with high-throughput single-cell RNA-sequencing, where iterative rounds of cell labeling enable the construction of multi-level lineage trees. This straightforward lentiviral-labeling approach can be applied to an array of cell biological applications to simultaneously profile lineage and identity, at single-cell resolution.
Proper citation: Brent A. Biddy, Wenjun Kong, Kenji Kamimoto, Chuner Guo, Sarah Waye, Tao Sun, Samantha Morris 2019. Single-cell mapping of lineage and identity via CellTagging. protocols.io dx.doi.org/10.17504/protocols.io.yxifxke Copy
Authors: Nathan Lee
Group: SPARC
Proper citation: Nathan Lee 2020. Abdominal Emitter Implantation. protocols.io dx.doi.org/10.17504/protocols.io.bpzbmp2n Copy
Authors: Amy Lyden, Gloria Castañeda, Emily Crawford, Saharai Caldera, Jenai Quan, Katherine Travisano
Group: Chan Zuckerberg Biohub
Summary: FLASH is a crispr-cas9 technology that enriches for targeted sequences in sequencing libraries. The initial DNA sample undergoes a blocking step that removes of the 5’ phosphoryl groups of the DNA fragments, resulting in a product that is not amendable for downstream adaptor ligation or amplification via standard Illumina-based library preparation. The subsequent incorporation of targeted CRISPR-cas9 library exposes the desired regions of interest, allowing them to be processed into a library. For more information on methods and results, please see the FLASH paper. For FLASH guide RNA design help, please see our github.
Proper citation: Amy Lyden, Gloria Castañeda, Emily Crawford, Saharai Caldera, Jenai Quan, Katherine Travisano 2019. FLASH v2.0. protocols.io dx.doi.org/10.17504/protocols.io.6iwhcfe Copy
Authors: Sangderk Lee
Group: Sangderk Lee lab
Proper citation: Sangderk Lee 2017. Lipoprotein Cholesterol Distribution Assay by FPLC. protocols.io dx.doi.org/10.17504/protocols.io.izhcf36 Copy
Authors: Sarah Giuliani
Group: Coleman Lab
Summary: Purpose: To concentrate harvested phage lysate up to 20X - 40X. The Vivaflow device uses a peristaltic pump setup to recirculate the input sample over a 100,000 MWCO PES membrane, which allows the media filtrate to be diverted and the phage to remain in the starting sample.
Proper citation: Sarah Giuliani 2016. Concentration of Phage Lysate using Vivaflow Tangential/Crossflow Filtration Cassette. protocols.io dx.doi.org/10.17504/protocols.io.fhdbj26 Copy
Authors: Bonnie Hurwitz and Murat Meren
Group: Hurwitz Lab, MetaFunc Course
Summary: This protocol describes basic functions to view your contigs and profiles databases in Anvi'o.
Proper citation: Bonnie Hurwitz and Murat Meren 2016. MG_HW12: Anvi'o interactive. protocols.io dx.doi.org/10.17504/protocols.io.gg8btzw Copy
Authors: Linbin Huang, Xinghong Yan
Summary: Pyropia yezoensis is one of the most valuable and widely cultivated seaweeds across the world. However, there is limited data regarding its genetic background underlying complex economic traits. The molecular genetic linkage map of P. yezoensis was not constructed. The most probable cause was the lack of mapping population, which was the basic of linkage analysis. In the present study, a red-type and fast-growing pigmentation mutant Py-HT and a wild-type strain Py-LS who had enough variation for traits of interest at both DNA and phenotypic level were crossed under control. The heterozygote (heterozygous conchocelis) was identified among conchocelis colonies which were developed from single zygotospore released from zygotosporangia of fertilized Py-HT blade, based on the result that 91.9% F1 gametophytic blades developed from the conchospores of heterozygous conchocelis were linearly sectored with 2-4 color sectors of parental colors. Afterwards, 57 chimeric blades with four color sectors were screened from thousands of F1 blades and 228 color sectors were separated according the boundaries of color sectors for single culture. A single zygotospore released from zygotosporangia of one sector through selfing was selected and then developed into homozygous conchocelis, which was declared a double haploid strain. Finally, a mapping population with 148 strains obtained from 37 of the 57 four color sectored blades was developed, which was used for the construction of genetic linkage maps and analysis of quantitative trait loci of P. yezoensis blades.
Proper citation: Linbin Huang, Xinghong Yan 2019. Development of a doubled haploid mapping population of Pyropia yezoensis and measurement of the economic characters of blade. protocols.io dx.doi.org/10.17504/protocols.io.x3vfqn6 Copy
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