bluuebunny/crossref_metadata_2025 huggingface dataset:
PUBERTY OF TWO SPECIES OF FLOUNDER (PLEURONECTES QUADRITUBERCULATUS AND LIMANDA ASPERA) (PLEURONECTIDAE) OFF THE COASTS OF SOUTH-EASTERN KAMCHATKA
Authors:   Kamchatka Branch of the All-Russian Research Institute of Fisheries and Oceanography (KamchatNIRO), Ovcherenko R.T.
Published: May 2022

The paper presents the characteristics of sexual maturation of the yellowfin sole Limanda aspera and the lemon sole Pleuronectes quadrituberculatus off the coast of southeastern Kamchatka in 1959–2020. The proportion of mature fish depending on the length was analyzed on the basis of empirical and theoretical data, the dynamics of maturation of the gonads of males and females of two flounders by months was characterized. In the Pacific waters of Kamchatka, males and females of the yellowfin sole mature at a smaller size than in the eastern part of the Bering Sea and at a larger size than off the coast of Western Kamchatka. Mass maturation of lemon sole takes place first in the eastern part of the Bering Sea, then near southeastern Kamchatka, and lastly on the western Kamchatka shelf.
POSSIBILITIES OF USING BROWN SEAWEEDS IN BIOGAS PRODUCTION
Authors:   RUDN University, Shushpanova D.V. , Kapralova D.O. ,   RUDN University
Published: May 2022

It is proposed to use seaweeds of the order Laminariales growing in the waters of the White, Barents and Okhotsk seas for biogas production as an alternative fuel. A preliminary assessment of the life cycle of biogas production from brown seaweeds, including the use of substandard raw material or seaweeds from storm emissions has been carried out. The obstacles to their use from the point of view of economics, legislation and logistics are indicated.
[RETRACTED] Labelled Building Blocks v1
Authors: Sciences BOC
Published: May 2022

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Isotope labeled building blocks refer to replacing one or more atoms in building blocks molecular with stable isotopes. Stable isotope labeled building blocks have the advantages of high sensitivity, simple location and accurate quantification. Therefore, stable isotope labeled building blocks are widely used in the field of life science and environment. Application Fields Application in Biochemical Field In the field of biomedicine, stable isotope labeled building blocks could be used to overcome signal degradation during detection of molecule structures by NMR. NMR spectroscopy is an effective means to study the structure and dynamics of building blocks in solution at atomic resolution. However, building blocks analysis by NMR suffers from spectral overlap and line broadening. Isotope labeled building blocks could avoid these questions. Application in Environment Isotope labeled building blocks could be used to reveal microbially mediated carbon flow and carbon cycling processes. Isotope-labeled building blocks could indicate the types of carbon sources utilized by microorganisms and the means by which they assimilate these carbon sources, thus providing information on microbially mediated carbon flow and carbon cycling processes, and realizing the relationship between specific microbial communities and the carbon cycle they participate in.
SCOT – Short Course Oncology Therapy – A Study of Adjuvant Chemotherapy in Colorectal Cancer v1
Authors: Iveson Timothy
Published: May 2022

Design: Phase III, randomised controlled, two arm, open label, multicentre, non-inferiority. Objectives: Assessment of the efficacy of 12 weeks versus 24 weeks of treatment and associated toxicity. Economic analysis of the cost effectiveness of the 2 arms. Comparison of 2 randomisation methodologies. Endpoints: Primary Endpoint: • Disease free survival Secondary Endpoints: • Overall survival • Cost-effectiveness • Toxicity • Quality of Life Study population: 6087 patients with fully resected high-risk stage II or fully resected stage III colorectal cancer.
[RETRACTED] https://www.jpost.com/promocontent/keto-extreme-fat-burner-south-africa-price-at-clicks-dischem-denmark-living-lifestyle-705766 v1
Authors: not provided ketoextremefatburnerCOST
Published: May 2022

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Keto Extreme Fat Burner requires some genuine festival now and can be bought without a doubt in the least demanding ever way.
[RETRACTED] Enzymatically Cleavable Linkers v1
Authors: Sciences BOC
Published: May 2022

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Enzyme-cleavable linkers have emerged as a particularly effective ADC linker type due to their ability to selectively release payloads in the lysosomes of target cells. BOC Sciencesiscommittedto providing flexible ADC enzymatically cleavable linkers at competitive prices. In BOC Sciences, we support various ADC linkers development and bioconjugation services for global customers to achieve new drug discovery milestones with comprehensive and advanced platforms.
[RETRACTED] Total Peptide Library Construction Technology v1
Authors: Miller Cathy
Published: May 2022

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The total peptide libraryis a collection of a large number of small peptide of specific length and different sequences, which includes the permutation and combination of various amino acid sequences in the short peptide of this length. The general synthetic peptide library uses 20 kinds of natural amino acids as building units, and the size of the library capacity is determined by the number of amino acids (n) that constitute a peptide of a specific length, which can be simply calculated as 20n. For example, to construct a dipeptide library, there are 202(400=4×102) combinations, the tripeptide library has 203(8,000=8×103) combinations, and so on. Therefore, the larger library capacity complicates the synthesis of peptide drugs and becomes a bottleneck in the development of peptide drugs. Creative Peptides Design PlatformTM(CPDP) has developed peptide information compression technology, which uses bioinformatics methods to compress peptide information, which can integrate the information of multiple peptide into one peptide, so as to contain a large amount of peptide information in a relatively small storage volume. It opens up a new way to research and develop peptide drugs. Based on this technology, we have constructed a total peptide library, containing nearly 500 million different peptide sequence information in a library of approximately 80,000 peptides, which will increase the screening efficiency by approximately 6,000 times.
[RETRACTED] Labelled APIs v1
Authors: Sciences BOC
Published: May 2022

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Isotope-labeled API is a chemical reagent that contains one or several isotopic atoms in the molecule, and uses its traceability for analytical determination. The chemical properties of the reagents replaced by isotopes are usually unchanged except for isotopic effects. Therefore, isotope-labeled reagents can participate in similar chemical reactions and are widely used as tracers to study the laws of substance movement and change.
[RETRACTED] PNA-lipophilic Ligand Conjugates Synthesis v1
Authors: Miller Cathy
Published: May 2022

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Lipophilic ligands are a class of substances suitable for cell transport, they are lipids with a certain hydrophilic-lipophilic balance. When transported across the cell membrane, the hydrophilic end faces the extracellular water-soluble environment, and the hydrophobic end faces the cell membrane. A typical representative of this modification is the covalent attachment of bile acid and cholesterol to PNA. The conjugate also has a certain liver-targeting effect in vivo and has the potential to develop into a liver-targeted preparation. The covalent binding of PNA with hydrophobic compounds or lipids has some excellent properties. For example, by connecting a hydrophobic long carbon chain to the N-terminus of PNA and amino acid residues to the C-terminus, the conjugate has good hydrophilicity and lipophilicity, and shows better specificity when hybridizing with DNA. The conjugate can also self-assemble into spherical micelles, and has application potential in biosensors, gene drugs, etc. PNA-lipophilic ligand conjugate also shows good performance in cell transport. Creative Peptideshas completed many PNA conjugate synthesis cases. Based on these projects, our team has accumulated a deep understanding of product requirements. With rich experience and scientific knowledge, our experts are dedicated to helping our customers navigate and accelerate your PNA synthesis plan. By combining scientific knowledge and laboratory experience, we are committed to expanding your understanding and applications of PNA conjugates. Pleasecontact usto learn more.
Taxon group: Non-larval Arthropods (TSS1) v1
Authors: Pereira da Conceicoa Lyndall , Sivell Olga , Sivess Laura , Fletcher Chris , R. Broad Gavin , Crowley Liam , Januszczak Inez
Published: May 2022

This is part of the collection "DToL Taxon-specific Standard Operating Procedure (SOP) for the Terrestrial and Freshwater Arthropods Working Group". The SOP collection contains guidance on how to process the various terrestrial and freshwater arthropod taxa within the scope of the Darwin Tree of Life project. The guidance specifically refers to the tissue samples needed for DNA barcoding (which takes place at the Natural History Museum (NHM)) and outlines the dissected tissues required for whole genome sequencing (WGS), which takes place at the Wellcome Sanger Institute . Every specimen is submitted for DNA barcoding first before potentially being sent to the Wellcome Sanger institute. TSS1 = Taxon Specific SOP for Non-larval Arthropods Definition: Adult terrestrial and freshwater arthropods and immature life stages of hemimetabolous taxa; over 5 mm body length. Including: Coleoptera, Hymenoptera, Diptera, Lepidoptera, other insect orders (Blattodea, Dermaptera, Ephemeroptera, Hemiptera, Mecoptera, Megaloptera, Neuroptera, Odonata, Orthoptera, Plecoptera, Raphidioptera, Trichoptera), Amphipoda, Isopoda, Diplopoda, Chilopoda, Araneae, Opiliones, Scorpiones. Excluding: Specimens smaller than 5mm Pictorial dissection guides available. See the Guidelines for important details and checklist.
[RETRACTED] Peptide N-Terminal Modification v1
Authors: Miller Cathy
Published: May 2022

[RETRACTED]

Although many of the most widely recognized post-translational modifications are characteristic of secretory or cell-surface proteins, most proteins, whatever their ultimate cellular desination, undergo some modification. For proteins synthesized completely within the cytoplasm, the earliest and most widespread are removal or modification of the N-terminal residue. In many proteins the N-terminal α-ammonium group (PK=8) undergoes secondary modification. N-terminal modification reduces overall solubility of the peptide by reducing its overall charges. However, the stability of the peptide could also be increased because N-terminal modification generates a closer mimic of the native protein. Therefore, these modifications might increase the biological activity of a peptide and prevent degradation by enzymes. Creative Peptides is specialized in the custom synthesis of N-terminal modification peptides, providing a confidential and efficient service at competitive prices. Every step of peptide synthesis is subject to Creative Peptides' stringent quality control. Typical delivery specifications include HPLC chromatogram Mass spec analysis Synthesis report Certificate of analyses
Deleidi Lab | Protocols | "Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly v1
Authors: not provided michela.deleidi , José Pérez J. María , Raji Hariam , Bertoli Federico , Baden Pascale
Published: May 2022

Collection of protocols of Team Deleidi used in the publication: "Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly and function"
Intracellular recordings and post hoc immunofluorescence v1
Authors: M Gwynne Rachel , Koussoulas Katerina , Bornstein Joel
Published: May 2022

The following protocol was submitted on behalf of the authors from the Bornstein lab by the SPARC project. This protocol describes methods for standard intracellular recording from mouse myenteric neurons impaled with intracellular electrodes containing biocytin, followed by processing for immunohistochemistry to map the projections of the neurites of enteric neurons. Methods described in this protocol are adapted from decades of studies on guinea-pig enteric neurons and more recent analyses of mouse duodenal myenteric neurons and colonic submucosal neurons. The immunofluorescence is used to reveal either calretinin or neuronal nitric oxide synthase (nNOS), as well as the enteric neurons in the proximal colon of the mouse. The methods can be generalized to whole mount preparations from any gut region in any species. Mice were sacrificed by cervical dislocation, a procedure approved by the University Melbourne Animal Experimentation Ethics committee. The experimental procedures should incorporate all local requirements for standards of animal experimentation.
Inducing gemmulation in the freshwater spongeEphydatia muelleriin culture using theophylline v1
Authors: Sogabe Shunsuke
Published: May 2022

Freshwater sponges produce overwintering cysts called gemmules that are full of stem cells, allowing them to survive harsh winter months. The gemmules of Ephydatia muelleri can be kept in 3°C for months, and in -80°C for years, while still remaining viable to hatch and develop into a functional sponge, as outlined in the protocol: "Hatching and freezing gemmules from the freshwater sponge Ephydatia muelleri" (https://dx.doi.org/10.17504/protocols.io.863hzgn). With a recently published chromosome-level assembly of the genome (Kenny et al., 2020), along with multiple transcriptomes, this makes Ephydatia muelleri an ideal sponge species to establish as a model sponge species that can be used in labs world-wide. While the gemmules used in the lab are usually collected from rivers and lakes every season for practical reasons, there have been studies showing the gemmulation process can be induced. A classic study looking at this process was conducted by Rasmont in 1974, who used theophylline to induce gemmulation in the freshwater sponge Ephydatia fluviatilis. Using up to 400uM of theophylline, this study showed that gemmulation can be induced in 100% of the sponges used in the experiments, and can occur as fast as 4 days after treatment with theophylline. While this is an interesting and important process to understand the biology of freshwater sponges, the ease and practicality of collecting masses of gemmules from wild populations most likely led to this process being rarely revisited in the lab. Here, we outline a protocol for inducing gemmulation in the freshwater sponge Ephydatia muelleri using theophylline, establishedby revisiting the study done by Rasmont (1974). In E. muelleri, the gemmulation process takes longer than in E. fluviatilis, with the fastest documented gemmulation detected at 8 days after treatment with theophylline, and taking up to 12+ days in some cases. The gemmulation process usually completes within 4-5 days once the gemmulation process can be detected, and results in a smaller gemmule size than the ones obtained from wild populations. The newly formed gemmules take roughly 6-7 weeks to mature in the 3°C incubator before they can be plated to hatch. While this is a lengthy process, being able to induce gemmulation in the lab increases the potential for using E. muelleri in transfection studies. References Kenny, N.J., Francis, W.R., Rivera-Vicéns, R.E. et al. Tracing animal genomic evolution with the chromosomal-level assembly of the freshwater sponge Ephydatia muelleri. Nature Communications. 2020. 11, 3676. https://doi.org/10.1038/s41467-020-17397-w. Rasmont R. Stimulation of cell aggregation by theophylline in the asexual reproduction of fresh-water sponges (Ephydatia fluviatilis). Experientia. 1974. Jul 15;30(7):792-4. doi: 10.1007/BF01924190. PMID: 4367998.
Quantitative detection of vitamin B12in algae by bioassay and ICP-MS/MS v1
Authors: Dupuis Sunnyjoy , Schmollinger Stefan , S. Merchant Sabeeha
Published: May 2022

This protocol describes two methods for determining the amount of vitamin B12 present in the spent medium and cell lysate of algae cultures. The first method is a bioassay, adapted from Mok, Hallberg, & Taga (2022), which estimates the B12 concentration in solution from the growth of a B12-requiring Escherichia coli mutant. The second method uses the direct detection of cobalt via Inductively Coupled Plasma Mass Spectrometry (ICP-MS/MS) as a proxy for vitamin B12. We describe the preparation of spent medium and cell extract fractions from the chlorophyte alga Chlamydomonas reinhardtii for each method, preparation of standard cyanocobalamin solutions, and the correlation between cobalt and cyanocobalamin in algal cells. We thank Michi Taga and Alison Smith for their guidance in optimizing the bioassay for C. reinhardtii.
Collecting Spleens for genotyping rats v1
Authors: J Barrero Gabriel , Polesskaya Oksana , Palmer Abraham
Published: May 2022

This protocol describes how to dissect and collect soft tissue (spleen) from rats. Collected spleen tissue is intended to be used for DNA extraction and genotyping
P1 v1
Authors: not provided maria
Published: May 2022

Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Pellentesque arcu. Phasellus faucibus molestie nisl. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? Suspendisse sagittis ultrices augue. Vestibulum erat nulla, ullamcorper nec, rutrum non, nonummy ac, erat. Nullam justo enim, consectetuer nec, ullamcorper ac, vestibulum in, elit. In convallis. Mauris elementum mauris vitae tortor. Sed ac dolor sit amet purus malesuada congue. Etiam ligula pede, sagittis quis, interdum ultricies, scelerisque eu. Nunc auctor. Nam sed tellus id magna elementum tincidunt. Sed convallis magna eu sem. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In dapibus augue non sapien.
P3 v1
Authors: not provided maria
Published: May 2022

Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Pellentesque arcu. Phasellus faucibus molestie nisl. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? Suspendisse sagittis ultrices augue. Vestibulum erat nulla, ullamcorper nec, rutrum non, nonummy ac, erat. Nullam justo enim, consectetuer nec, ullamcorper ac, vestibulum in, elit. In convallis. Mauris elementum mauris vitae tortor. Sed ac dolor sit amet purus malesuada congue. Etiam ligula pede, sagittis quis, interdum ultricies, scelerisque eu. Nunc auctor. Nam sed tellus id magna elementum tincidunt. Sed convallis magna eu sem. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In dapibus augue non sapien.
P3v2 v2
Authors: not provided maria
Published: May 2022

Lorem ipsum dolor sit amet, consectetuer adipiscing elit. Pellentesque arcu. Phasellus faucibus molestie nisl. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? Suspendisse sagittis ultrices augue. Vestibulum erat nulla, ullamcorper nec, rutrum non, nonummy ac, erat. Nullam justo enim, consectetuer nec, ullamcorper ac, vestibulum in, elit. In convallis. Mauris elementum mauris vitae tortor. Sed ac dolor sit amet purus malesuada congue. Etiam ligula pede, sagittis quis, interdum ultricies, scelerisque eu. Nunc auctor. Nam sed tellus id magna elementum tincidunt. Sed convallis magna eu sem. Quis autem vel eum iure reprehenderit qui in ea voluptate velit esse quam nihil molestiae consequatur, vel illum qui dolorem eum fugiat quo voluptas nulla pariatur? In dapibus augue non sapien.
Unilateral intranigral AAV alpha synuclein mouse model v1
Authors: Srivastava Pranay
Published: May 2022

This protocol details methods for the unilateral intranigral AAV alpha-synuclein injection
Striatal dopamine measurement through HPLC v1
Authors: Srivastava Pranay
Published: May 2022

Protocol for striatal dopamine measurement in mouse brain
Preparation of Biological Tissues for Serial Block Face Scanning Electron Microscopy (SBEM) v1
Authors: J. Deerinck Thomas , A. Bushong Eric , H. Ellisman Mark , Thor Andrea
Published: May 2022

This protocol was designed to enhance signal for backscatter electron imaging of epoxy embedded mammalian tissue at low accelerating voltages (1-3 keV). However, it can easily be adapted for use with tissues from other species, tissue culture cells, plants and microbial cells by adjusting the buffer strength and the duration of relevant steps. This combinatorial heavy metal staining protocol employs a battery of contrasting steps after primary aldehyde fixation including: ferrocyanide reduced osmium tetroxide post fixation, thiocarbohydrazide-osmium liganding (OTO) and subsequent uranyl acetate and en bloc lead aspartate staining. Calcium chloride is included in a number of steps to enhance membrane preservation and staining. This protocol was designed primarily to emphasize the contrast of membranes. Many other contrasting agents may be included to increase staining of other cellular and extracellular constituents.
Protein purification v1
Authors: not provided patreece.suen
Published: May 2022

This protocol details how to purify 3xFLAG-SHIP164Δ901-1099
Adaption for small samples - Nuclei isolation from snap-frozen tissue for single nucleus RNA Sequencing v1
Authors: Y Mimpen Jolet , Paul Claudia , Seed Network Tendon , Cribbs Adam , Snelling Sarah
Published: May 2022

Previously, we described a robust protocol for the isolation of nuclei from snap frozen fibrous tissues, including tendon and ligament, for use in single nucleus RNA Sequencing. This use of snap-frozen tissue enables tissues that are collected from different centres or/and over a period of time to be processed together. Therefore, this is not only convenient for prevention of a stress response and for collecting and processing tissue (especially tissues that are not very accessible), but it might also reduce batch effects by preventing the need to process tissues fresh on different days. The collection of (healthy) human tissues is difficult, meaning that there are often only small pieces available for research. Therefore, we present an adapted protocol for the isolation of nuclei for single nucleus RNA sequencing from small pieces of tissue (biopsies and tissues up to ~100mg). This protocol makes use of smaller tubes and small volumes of buffers, thereby reducing nuclei loss through transfer steps, leading to great nuclei yields per tissue weight. This protocol was adapted from Slyper et al. (2020) Nature Medicine.
LRRK2 microtubule sedimentation binding assay v1
Authors: Dickey Andrea
Published: May 2022

Assay to determine LRRK2 protein binding to microtubules. Original assay by Andrea Dickey. Adapted by Mariusz Matyszewski for protocols.io. Assay originally used in Snead, Matyszewski, Dickey et al. 2022
TMR labeling of LRRK proteins v1
Authors: Snead David
Published: May 2022

Protocol for non-specific TMR labeling of LRRK1 and LRRK2 RCKW proteins. Protocol developed by David Snead and adapted by Mariusz Matyszewski for protocols.io. Written as used in Snead, Matyszewski, Dickey et al. 2022.
Volume Endoscopic Procedures and COVID-19 Cases v1
Authors: C. Sjaaf Amal , Achadi Anhari , Soewondo Prastuti , Rosnim Roswin , A. Gani Rino , Olivia Maurine Jasirwan Chyntia
Published: May 2022

The impact of the COVID-19 pandemic causedthe decreasing ofhealthcare services,theintervention of non-surgical procedures, and endoscopy. This study examined the volume of endoscopy at Dr. Cipto Mangukusumo Hospital (RSCM)as the highest referral hospital in Indonesia. A cross-sectional mixed-method was used to assess the relationship between endoscopy volume, age, gender, number of COVID-19 cases, type ofpatient’scase, the origin of treatment, and kind of endoscopic procedure before and during the pandemic. This period was divided into six intervals of three months each, respectively,from January 2020 to September 2021, and 5,030 endoscopic procedures were collected. There was a significant difference in the volume of endoscopes before and during the pandemic, specifically at the early stage when preparations were not optimal. Furthermore, there was a high wave of Delta Variant cases from May to July 2021.In addition, thefactors of age, type ofpatient’scase, origin, and type of treatment showed significant differences before and during the COVID-19 pandemic.Finally,changes in the flow of servicesalsoinfluenced other various impacts in endoscopy and service costs. Therefore, further study is required to calculate the unit costs.
Safety and efficacy, exercise-based cardiac rehabilitation for patients with atrial fibrillation after catheter ablation therapy: a protocol for a systematic review and meta-analysis. v1
Authors: KURASAWA Yasuyuki , Maeda Hirotada , Tamaru Tetsuro , Sasaki Yukihiro , Matsumori Keishi , Yokokawa Yoshiharu , Kitagawa Takashi
Published: May 2022

This review aimed to assess whether exercise-based cardiac rehabilitation for patients with atrial fibrillation after catheter ablation can be safely implemented and contribute to maintaining sinus rhythm, patient exercise function, and quality of life. We plan to include all randomized controlled trials (RCTs) of cardiac rehabilitation designed to improve motor function in patients with post-catheter ablation AF patients. (P: AF patient after catheter ablation therapy (Adult patients 40 years and older), I: Exercise-based cardiac rehabilitation, C: usual care, O: Serious adverse events (recurrence of AF, death, rehospitalization), Quality of life, exercise capacity, cardiac function . In this review, we plan to use five databases. Two reviewers will screen articles to identify studies for inclusion. We will use the Risk of Bias 2 tool for risk of bias assessment.
2022 GenomeTrakr Proficiency Testing exercise (PulseNet Harmonized) v5
Authors: Balkey Maria , Timme Ruth , Haendiges Julie
Published: May 2022

This SOP outlines guidelines on how to process the isolates for the 2022 GenomeTrakr (GT) Proficiency Testing exercise. This SOP is applicable to all GenomeTrakr labs participating in the 2022 GenomeTrakr Proficiency Testing exercise (PulseNet Harmonized). The FDA GenomeTrakr program will ship the following proficiency testing isolates on April, 2022. ESP22-5191 Shigella SAP22-0236 Salmonella SAP22-0692 Salmonella SAP22-1300 Salmonella Completion of the entire proficiency test entails the following: GT participating laboratory generates sequencing data (fastq files) using the PT strains provided by CDC through FDA-CFSAN-WGS Program-GT in accordance to GT and/or PulseNet SOPs. Populate sample sheet according to 2022 GT Proficiency Testing exercise (PulseNet Harmonized) SOP Submission of sequencing records to the appropriate project on BaseSpace or Isilon according to GT SOPs By participating in the 2022 GT Proficiency Testing exercise (PulseNet Harmonized), GT labs provide consent to use the PT exercise data in subsequent analysis and manuscript publications. Participants will be acknowledged for their contribution on any publication that might require processing data from the 2022 GT PT exercise. v5: Added links to GalaxyTrakr_PT_exercise_report_2022.xlsx
RasberryPi-computer based phenotyping for side view image process v2
Authors: Yu Li'ang , M Julkowska Magdalena
Published: May 2022

Imaging of plants can be done in an inexpensive way, using Raspberry Pi based setup. Here - we describe how we are processing the side-view images taken from 7 different sides - using the PlantCV pipeline.
RatGTEx pipeline v1
Authors: Munro Daniel , M Saba Laura , Chen Hao , Palmer Abraham , Mohammadi Pejman
Published: May 2022

This is the pipeline used to process data for the RatGTEx Portal. It is loosely based on the GTEx eQTL mapping pipeline, though it includes some utility scripts from there in their entirety. All code for this pipeline can be found in the repository. It is built on Snakemake, a Python-based framework for reproducible data analysis. The commands reproduced here use Snakemake-style templating, with variables in brackets to represent different input/output file names and parameters.
Testing 1, 2 v1
Authors: not provided shanley
Published: May 2022

Testing, testing.
RT-qPCR Detectionof SARS-CoV-2 from Wastewater Using the AB 7500 v2
Authors: not provided Jacquelina.Woods , not provided rachel.rodriguez
Published: May 2022

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol describes triplex and duplex assays for the RT-qPCR detection of the nucleocapsid region of the SARS-CoV-2 genome. These assays, along with the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay (RT-qPCR Detectionof Process Controls (Murine noroviurs and crAssphage) from Wastewater (protocols.io)), were developed for use on the AB 7500 platform using software version 2.0 or 2.3. All assays incorporate an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. These multiplexed detection assays were developed for the qualitative determination SARS-CoV-2 nucleocapsid gene extracted from wastewater. Valid sample results are contingent upon the detection of the MNV extraction control from the sample being tested.
RT-qPCR Detectionof Process Controls (Murine noroviurs and crAssphage) from Wastewater using AB 7500 v2
Authors: not provided Jacquelina.Woods , not provided rachel.rodriguez
Published: May 2022

This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This protocol describes the murine norovirus (MNV; extraction control) and crAssphage (human indicator) RT-qPCR assay developed for use on the AB 7500 platform using software version 2.0 or 2.3. The assay incorporates an internal amplification control (IC) to prevent the reporting of false negatives due to inhibition or failure of the RT-qPCR. This multiplexed detection assay was developed for the determination crAssphage extracted from wastewater, as an endogenous control, and MNV as an extraction control. The assay is designed to be used in conjunction with the SARS-CoV-2 RT-qPCR detection assay. Valid sample results for SARS-CoV-2 detection are contingent upon the detection of the MNV extraction control from the sample being tested.
Stranded Transcript Count Table Generation from Long Reads v13
Authors: A Eccles David
Published: May 2022

This protocol is for comparing different samples at the transcript level, using long reads that are mapped to transcripts. Input(s): demultiplexed and oriented fastq files (see protocol Preparing Reads for Stranded Mapping), transcript reference fasta file, annotation file Output(s): transcript table, sorted by differential coverage, annotated with gene name / description / location
Activation Induced Marker (AIM) Staining Protocol v1
Authors: P. Williams Gregory , not provided cecilia
Published: May 2022

This protocol details about activation induced marker staining.
SCA-seq v1
Authors: Tang Chong
Published: May 2022

methylation，cut and ligation
LCM-NanoPOTS workflow for spatial proteome mapping v1
Authors: Kwon Yumi , S Nakayasu Ernesto , QIAN WEI-JUN , Zhu Ying
Published: May 2022

Spatial proteomics holds great potential to transform our understanding of the role of cell populations and their spatial organizations in human tissues related to diseases. However, in-depth proteome measurement with high spatial resolution has been challenged by the low sample input and inefficient proteomics workflow. In this protocol, we outlined the detailed procedure for high-resolution analysis of human pancreas tissue sections using a spatial proteomic platform by combining laser capture microdissection (LCM), nanoPOTS (nanodroplet processing in one pot for trace samples), and nanoflow liquid chromatography mass spectrometry (LC-MS). This protocol has been applied to human pancreas specimens for HubMAP Tissue Mapping Centers. Expected outcome: The unbiased spatial proteome mapping of >3000 proteins at 50-µm spatial resolution depending on sample types and LC-MS instrumentation.
JAX - 10x Genomics - NEXTGEM Multiome ATAC + Gene Expression v1
Authors: F F Flynn William
Published: May 2022

Standard 10x Genomics - NEXTGEM Multiome protocol used by the JAX Single Cell Biology lab for the simultaneous single nucleus chromatin accessibility and gene expression assay.
JAX SCBL workflow for scRNA-seq of human placenta v1
Authors: Luo Diane , Grassmann Jessica , not provided Paul.gabriel , F F Flynn William , Courtois Elise , Robson Paul
Published: May 2022

Described here is the workflow used by the JAX Single Cell Biology lab to generate single cell transcriptomic libraries from human placenta samples collected at the UCSD HuBMAP Female Reproductive Tissue Mapping Center.
Automatic Multi-functional Integration Program (AMFIP) towards All-optical Mechano-electrophysiology Interrogation v1
Authors: Luo Qin
Published: May 2022

Automatic operations of multi-functional and time-lapse live-cell imaging are necessary for the biomedical science community to study active, multi-faceted, and long-term biological phenomena. To achieve automatic control, most existing solutions often require the purchase of extra software programs and hardware that rely on the manufacturers’ own specifications. However, these software programs are usually non-user-programmable and unaffordable for many laboratories. To address this unmet need, we have developed a novel open-source software program, titled Automatic Multi-functional Integration Program (AMFIP), as a new Java-based and hardware-independent system that provides proven advantages over existing alternatives to the scientific community. Without extra hardware, AMFIP enables the functional synchronization of the μManager software platform, the Nikon NIS-Elements platform, and other 3rd party software to achieve automatic operations of most commercially available microscopy systems, including but not limited to those from Nikon. AMFIP provides a user-friendly and programmable graphical user interface (GUI), opening the door to expanding the customizability for myriad hardware and software systems according to user-specific experimental requirements and environments. To validate the intended purposes of developing AMFIP, we applied it to elucidate the question whether single cells, prior to their full spreading, can sense and respond to a soft solid substrate, and if so, how does the interaction depend on the cell spreading time and the stiffness of the substrate. Using a CRISPR/Cas9-engineered human epithelial Beas2B (B2B) cell line that expresses mNeonGreen2-tagged mechanosensitive Yes-associated protein (YAP), we show that single B2B cells develop distinct substrate-stiffness-dependent YAP expressions within 10 hours at most on the substrate, suggesting that cells are able to sense, distinguish, and respond to mechanical cues prior to the establishment of full cell spreading. In summary, AMFIP provides a reliable, open-source, and cost-free solution that has the validated long-term utility to satisfy the need of automatic imaging operations in the scientific community.
FLASH-seq UMI protocol v2
Authors: Picelli Simone , Hahaut Vincent
Published: May 2022

The single-cell RNA-sequencing (scRNA-seq) field has evolved tremendously since the first paper was published back in 2009. While the first methods analysed just a handful of cells, the throughput and performance rapidly increased over a very short timespan. However, it was not until the introduction of emulsion droplets methods, that the robust and reproducible analysis of thousands of cells became feasible. Despite generating data at a speed and a cost per cell that remains unmatched by full-length protocols like Smart-seq, scRNA-seq in droplets still comes with the drawback of addressing only the terminal portion of the transcripts, thus lacking the required sensitivity for comprehensively analyzing the transcriptome of individual cells. Building upon the existing Smart-seq2/3 workflows, we developed FLASH-seq (FS), a new full-length scRNA-seq method capable of detecting a significantly higher number of genes than both previous versions, requiring limited hands-on time and with a great potential for customization.
FLASH-seq Low-Amplification protocol v2
Authors: Picelli Simone , Hahaut Vincent
Published: May 2022

Building upon the existing Smart-seq2/3 workflows, we developed FLASH-seq (FS), a new full-length scRNA-seq method capable of detecting a significantly higher number of genes than both previous versions, requiring limited hands-on time and with a great potential for customization. FLASH-seq Low-Amplification (FS-LA), represents FS quickest iteration, generating sequencing-ready libraries in 4.5 hours by removing intermediate cleanups and QC steps and without sacrificing performance. FS-LA is the best choice when a large number of plates need to be processed in parallel.
Stranded Mapping from Oriented Long Reads v9
Authors: A Eccles David
Published: May 2022

This protocol demonstrates how to map strand-oriented long reads to a genome, and visualise them in a genome browser. The general idea is to use minimap2 to create stranded BAM files, which are split for forward/reverse orientation then converted into BigWig format for display in a genome browser. Input(s): stranded fastq files (see protocol Preparing Reads for Stranded Mapping) a FASTA file containing the genome / sequence of interest. Output(s): Genome-mapped stranded BAM files Genome-mapped stranded BigWig files
Workflow for human placental bulk ATACseq v1
Authors: Lindsay-Hewett Scott , Stanley Valentina , Parast Mana , Laurent Louise
Published: May 2022

Described here is the workflow used by the Female Reproductive Tissue Mapping Center at UCSD to generate bulk ATACseq data from human placenta.
Plant assemble - Plant de novo genome assembly, scaffolding and annotation for genomic studies v1
Authors: Ferguson Scott , Jones Ashley , Borevitz Justin
Published: May 2022

With the advancement of long-read sequencing technologies and associated bioinformatics tools, it has now become possible to de novo assemble complex plant genomes with unrivalled contiguity, completeness and correctness. As read lengths can surpass repeat lengths, the ability to assemble genomes de novo has dramatically improved, whereby complex plant genomes of widely variable sizes and repeat content have highly benefited. Despite these improvements, challenges remain in performing de novo assembly, namely in developing a reliable workflow and in tool choice. Here we present a protocol collection of bioinformatic workflows detailing plant genome assembly using Oxford Nanopore Technologies long-reads with a de novo assembler (Canu), syntenic or Hi-C scaffolding, and RNA and/or gene homology-based annotation. We have developed and tested these protocols on multiple plant genomes. Using these protocols with sufficient coverage of long-reads, a highly contiguous, complete, and correct plant genome can be assembled. These genomes can further genomic research into structural variation among groups, and SNP genotyping and association studies among populations.
Automated DNA template preparation and quantitation methods v1
Authors: Romantseva Jane , Ross David , Alperovich Nina , Strychalski Elizabeth
Published: May 2022

Concentrated, intact, and biologically-active DNA templates are essential for protein production using cell-free expression. We present automated methods for DNA template preparation and quantitation towards improving reproducibility in cell-free expression.
Detection and Quantification of Calcitonin Gene-Related Peptide in Human Blood Plasma using a Modified Enzyme-Linked Immunosorbent Assay v1
Authors: S. Krishnan Pavan , T. Zamuner Fernando , M. Jenks Carolyn , Xie Yanjun , Zhang Lisa , Lehar Mohammed , Iglesias Lozano Marcos , S. Fedarko Neal , R. DeVine Lauren , Brait Mariana , N. Cole Robert , P. Carey John
Published: May 2022

Calcitonin gene-related peptide (CGRP) is a vasoactive neuropeptide that plays a putative role in the pathophysiology of migraine headaches and may be a candidate for biomarker status. Here, we present a protocol for purification and quantification of CGRP in human plasma. This protocol involves sample collection and preparation, extraction as a means of purification, and quantification via enzyme-linked immunosorbent assay (ELISA). After finding unrealistically high CGRP concentrations in spike-and-recovery experiments using a commercial CGRP ELISA kit protocol, we modified the existing protocol to produce satisfactory percent recoveries. This protocol can theoretically be used to quantify CGRP concentrations in plasma of individuals not only with migraine, but also with other diseases in which CGRP may play a role.
Measurement of pancreatic islet beta-cell proliferation by flow cytometry v1
Authors: Ghislain Julien , Poitout Vincent
Published: May 2022

This protocol describes the steps to assess pancreatic beta-cell proliferation by flow cytometry. It is suitable for islets isolated from both rodents and humans. We routinely apply this protocol to quantify total and proliferating beta-cells but, with the appropriate antibody-fluorophore conjugates, can be easily adapted to assess other islet cells types (eg. alpha and delta) and markers (eg. apoptosis). Briefly, islets labelled with the proliferation marker EdU are dispersed, dead cells labeled and fixed. EdU is detected using the Click-iT Plus EdU Flow Cytometry Assay Kit and beta-cells immunostained with fluorophore-coupled anti-insulin antibodies. Beta-cell proliferation is then determined by flow cytometry by calculating the percentage of double-positive cells for EdU and insulin over the corresponding total insulin-positive cell population.
Measuring pattern separation in hippocampus by in situ hybridization v1
Authors: Eom Kisang
Published: May 2022

Pattern separation is a process that minimizes overlap between neuronal activity patterns those representing similar experiences. The mechanisms of pattern separation have been investigated for several decades. Theoretical models suggest that dentate gyrus (DG) segregates cortical input patterns before relaying its discriminated output patterns to CA3. So evaluation of neural ensembles in DG and CA3 have an important part for investigation of pattern separation. Measuring of entorhinal cortex (EC), DG and CA3 ensembles were largely depended upon the electrophysiological recordings, which is technically difficult. We present that more simple and convenient method for measuring pattern separation based on molecular method in this article.
Yeast Transformation Protocol v1
Authors: Nayak Sudhir
Published: May 2022

This protocol is based on Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. Methods in Enzymology 350: 87-96. Some steps of this protocol are based on information obtained from: http://www.umanitoba.ca/faculties/medicine/biochem/gietz/ This protocol is generalized and not to be considered optimal for all strains, conditions, and applications. Some aspects of this protocol may have to be modified significantly to suit your particular needs. It works for simple transformation, targeting in yeast, GAP repair, and multi-plasmid transformations for two-hybrid.
Volume Electron Microscopy Protocol for the ASP-1000 v1
Authors: S Stempinski Erin , Riesterer Jessica , Adamou Steven , S Lopez Claudia
Published: May 2022

Volume electron microscopy (vEM) requires a multitude of heavy metal stains to ensure adequate sample contrast and conductivity under the electron beam. Bench protocols for vEM can be time- and labor-intensive. The mPrep™ ASP-1000™ automated specimen processor equipped with a thermal control unit reduces the amount of time and labor required for vEM protocols.
Modified NEBNext® VarSkip Short SARS-CoV-2 Enrichment and library prep for Oxford Nanopore Technologies- adapted for wastewater samples v1
Authors: Ramachandran Padmini , Walsky Tamara , Windsor Amanda , Grim Chris , Hoffmann Maria
Published: May 2022

This protocol details methods for the preparation of SARS-CoV-2 sequencing library using VSS primers from library preparation from NEBNext® ARTIC SARS-CoV-2 Companion Kit (Oxford Nanopore Technologies®), NEB #E7660S/L 24/96 reactions adapted for wastewater samples. Standard Protocol with PCR Bead Cleanup: This protocol includes a cleanup and normalization step for each sample after cDNA synthesis. Performing the cleanup and normalization step creates library pools where the reads for each library are more evenly distributed. These pools will likely achieve sufficient and equal coverage in less run time, but they take more hands-on time This protocol also includes options for barcoding with dual indexing or single indexing of the samples.
Water filtration withZymo Bashing Bead tubes andDNA/RNA Shield reagent for subsequent DNA and RNA extraction from microbial communities v1
Authors: A Hempel Christopher
Published: May 2022

Filtration of water samples withZymo Bashing Bead tubes andDNA/RNA Shield reagent for subsequent DNA and RNA extraction from microbial communities.
Parallel DNA+RNA extraction from freshwater samples using the Quick-DNA/RNA Microprep Plus Kit and Zymo-Spin II-µHRC Filters (Zymo Research) v1
Authors: A Hempel Christopher
Published: May 2022

Parallel DNA+RNA extraction from freshwater samples using the Quick-DNA/RNA Microprep Plus Kit and Zymo-Spin II-µHRC Filters (Zymo Research) Note: maximum of 11 samples + extraction control possible at a time
Shipping wastewater samples to FDA-CFSAN v1
Authors: Balkey Maria
Published: May 2022

This SOP provides guidance for shipping wastewater samples to the US FDA Center for Food Safety and Applied Nutrition (CFSAN), covering specific steps for registering samples with CFSAN and for the preparation of shipments (documentation, packaging, notifications). We recommend that samples are shipped in two batches, one batch after 3 months of sampling and one batch at the conclusion of your sampling (~6 months).
protocol 2 v1
Authors: not provided richard
Published: May 2022

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Plant Chromatin Immunoprecipitation v1
Authors: Poza-Viejo Laura , del Olmo Ivan , Crevillén Pedro
Published: May 2022

Chromatin Immunoprecipitation (ChIP) is a crucial technique to study chromatin regulation, epigenetic phenomena and transcription factor DNA-binding in vivo. This technique is widely used in model plant systems like Arabidopsis but reliability in other plant systems is challenging. We adapted our well-established Arabidopsis ChIP protocol to be used with leaves and inflorescences of Brassica crops species (Brassica rapa and Brassica napus). This protocol is based on Gendrel et al. Nature Methods volume 2, pages 213–218 (2005), but has several modifications including chromatin sonication using a BioRuptor, immunoprecipitation using magnetic Dynabeads, and DNA elution is performed using Chelex 100 for ChIP-PCR or magnetic SPRI beads for ChIP-seq. The protocol is quick and very reproducible and has been validated against a number of histone modifications and protein tag antibodies. In this new version V2 we describe how to purify and recover DNA after immunoprecipitation if DNA is used for next-generation sequencing library preparation in ChIP-seq experiments.