bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced QIAseq DIRECT SARS-CoV-2 Kit for Illumina MiSeq v3
Authors: Ramachandran Padmini , Walsky Tamara , Windsor Amanda , Hoffmann Maria , Grim Chris
Published: May 2022

This SARS-CoV-2 targeted, a rapid viral RNA library preparation kit compatible with Illumina sequencers, provides an approximately five hour turn-around time from viral RNA sample to sequencing. The kit includes cDNA synthesis, SARS-CoV-2 enrichment, and library amplification and indexing reagents. The Enhanced protocol removes the need for normalization and quantification prior to amplification and indexing. RNA input is 5 μl, regardless of SC2 viral load in the sample extract. Amplification is performed using two primer pools that produce overlapping amplicons that are approximately 250 bp in size; as a result, no fragmentation is necessary prior to sequencing on Illumina platforms. 10 bp unique dual indexes (UDIs) are provided in the kit, and with a cycle read length of 149 for each paired-end read, the sequencing time is just over 28 hours. Version Updates: V2- Recommending 1:10 dilution of index primer on low viral titer samples to reduce short fragments. Controlled vocabulary for the use of positive control and negative template control has been added to the Sample Sheet following miseq loading guidelines.
Intracellular metabolomics extraction v1
Authors: Sanchez Juan
Published: May 2022

This quench and intracellular extraction are optimal for the analysis of metabolites stable at high temperatures and moderately acidic pH that are rapidly consumed such as pyruvate and acetyl-CoA. It also avoids the use of methanol which can form degradation products with many metabolites of interest. It provides a near-complete extraction of polar and semi-polar metabolites with negligible benefits from re-extraction. It also enables the simultaneous generation of intracellular and extracellular metabolomics samples amenable for stable isotope resolved metabolomics timecourse experiments. The compound classes extracted are best seen when paired with HILIC chromatography.
2022 GenomeTrakr Proficiency Testing exercise (PulseNet Harmonized) v1
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.
Prymnesium parvum isolation, DNA preparation and qPCR diagnostic v1
Authors: Anglès d'Auriac Marc , Dale Trine , Thaulow Jens , Hobæk Anders
Published: May 2022

Detection and quantification of many organisms that create problems in aquaculture is normally based on microscopical analysis. A disadvantage of the method is that it is time consuming especially if one needs a low limit of detection. When it comes to harmful algae, many species are small and fragile and are easily destroyed by conservation. In addition, many species are difficult to identify and requires highly qualified personnel. Over the last decade, however, there has been a rapid development of various qPCR based diagnostic tools, and although these have so far been used mostly for research purposes, several studies show that they can be used in more routine monitoring. The protocol described here enables isolation, DNA preparation and qPCR diagnostic of the harmful algae Prymnesium parvum.
How to select the best gRNA(s) for frameshift knockouts in zebrafish v1
Authors: Kroll Francois
Published: May 2022

Remember to cite the protocol if it is helpful to you! This protocol describes my approach (as of April 2022) to select gRNAs to generate frameshift knockouts in zebrafish using CRISPR-Cas9. It assumes you are selecting 3 gRNAs to generate F0 knockouts, following the method we described in doi: 10.7554/eLife.59683 and protocol 10.17504/protocols.io.bs2rngd6. Are you making a stable knockout line carrying a frameshift mutation? You can also follow this protocol to select a unique gRNA. Get in touch for questions/suggestions twitter –@francois_kroll email –francois@kroll.be
PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX) v2
Authors: Huang Yin-Tse
Published: May 2022

PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)
Stranded Transcript Count Table Generation from Long Reads v15
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
Mal-Herbal Protocol Version 8 dated 20th August 2020 v1
Authors: not provided kamakak
Published: May 2022

Background: Malaria is a disease of poverty and is still a major global health problem, especially in Africa. Almost all African countries are located in a malaria-endemic area (Figure 1). It was estimated that 228 million cases of malaria occurred worldwide in 2018.The development of insecticide and artemisinin resistance threatens the gains that have been made in the control and elimination of malaria. Tanzania is among the top ten highest-burden countries in the world. Therefore, the scientific investigation of antimalarial herbal remedies to prove their efficacy and safety may be of benefit to the worldwide agenda of malaria control and elimination.Objective: The first primary objective of this study will be to assess the safety and tolerability of malaria herbal remedy of Maytenus senegalensis among healthy male adults aged 18 to 45 years in Tanzania. The second objective is to evaluate the safety, tolerability as well as efficacy of malaria herbal remedy Maytenus senegalensis (MALHERBAL) for the treatment of Tanzanian adults aged 18 to 45 years with uncomplicated malaria compared to Artemether-lumefantrine.Materials & Methods: The study design of the trial will be single-center phase IIa and open-label clinical research with a population of 116 male participants aged 18 to 45 years residing in Bagamoyo District or nearby districts. The primary endpoint is the number of adverse events (AE) after drug intake until day 28. Secondary endpoints are the number of serious adverse events (SAE), adequate clinical and parasitological response rate (ACPR) on day 28, clinical and parasitological failure rates on days 3, 7, 14, and 28, changes in hemoglobin/hematocrit until day 28, and fever and parasite clearance time. Oocyst burden on days 7 and 14 as well as theNumber of developed early ring stage P. falciparum parasites. Case report form will be used to collect all study endpoint data. Categorical data will be presented by treatment group and time-point using counts and percentages of participants. Continuous variables will be also presented by treatment group and time-point, using (at least) the mean, standard deviation (SD), median, minimum (Min), maximum (Max), and number of observations (N). The analysis will be descriptive. In addition, 95% Confidence Intervals will be computed by the treatment group for all the efficacy end-points.
Plasmid-reprogramming of human fibroblasts v1
Authors: Baden Pascale , not provided michela.deleidi
Published: May 2022

This protocol details about plasmid-reprogramming of human fibroblasts.
X DOWNLOAD LG V20™ Software Update v1
Authors: not provided lg-
Published: May 2022

Verizon Wireless is pleased to announce a software update for your device. This software update has been tested to help optimize device performance, resolve known issues and apply the latest security patches.
HuBMAP | GE/ University of Washington Cell DIVE™ Modality Overview v1
Authors: McDonough Liz
Published: May 2022

This is an overview of all protocols currently in use for the GE/University of Washington Cell DIVE collaboration for the Human BioMolecular Atlas Program (HuBMAP). It includes links to each of the individual protocols that make up this project workflow.
Next Generation Sequencing of HIV-1 Drug Resistant Mutations v2
Authors: M McGruder Rawson Brenna
Published: May 2022

The Florida Department of Health's Bureau of Public Health Laboratories in Jacksonville has developed a protocol for the Next Generation Sequencing (NGS) of HIV, primarily for the purpose of drug-resistant mutation identification. This HIV-1 protocol uses amplicon-based sequencing based on primers designed by the BEEHIVE Consortium (https://www.beehive.ox.ac.uk/). The amplified pol gene regions can be used in both genotyping and drug resistance determination. Our protocol utilizes newer enzymes with higher fidelity for sequencing and Illumina sequencing technology. We have cross verified 3 different Illumina Sequencing platforms to ensure that all produce equivalent results so that in the event of a surge samples can be sequenced quickly and in mixed-species pools. The NGS data generated can also be used in surveillance and outbreak monitoring, giving epidemiologist more information about circulating viral genomes. There is also the potential that this protocol can be expanded to whole genome sequencing for HIV-1. The imminent sunsetting of ViroSeq (Abbott Molecular) has required many labs to look for new methods to continue identifying HIV-1 drug resistance strains for both clinical management and epidemiological study. NGS was chosen as it is more cost effective than investing in a single pathogen platform. NGS allows for one sample to produce results and data that can aid not just a patient but an entire population.
Coverage of open citations in DOAJ journals v1
Authors: Dami Constance , Bertozzi Alessandro , Manca Chiara , Kucuk Umut
Published: May 2022

This is the protocol for the research of the coverage of open citations in DOAJ journals. Our goal is to find out: about the coverage of articles from open access journals in DOAJ journals as citing and cited articles, how many citations do DOAJ journals receive and do, and how many of these citations involve open access articles as both citing and cited entities, as well as the presence of trends over time of the availability of citations involving articles published in open access journals in DOAJ journals.
Western blotting in Chlamydomonas reinhardtii v2
Authors: Vitor Molino João
Published: May 2022

This protocols describe the steps to perform a western blot in Chlamydomonas reinhardtii cell lysate and supernatant samples.
Mod3D Live Cell Chambers and holders 3D printing and Assembly v1
Authors: Ray Truant and
Published: May 2022

Live-cell microscopy imaging typically involves the use of high-quality glass-bottom chambers that allow cell culture, gaseous buffer exchange and optical properties suitable for microscopy applications. However, commercial sources of these chambers can add significant annual costs to cell biology laboratories. Consumer products in three-dimensional printing technology, for both Filament Deposition Modeling (FDM) and Masked Stereo Lithography (MSLA), have resulted in more biomedical research labs adopting the use of these devices for prototyping and manufacturing of lab plastic-based items, but rarely consumables. Here we describe a modular, live-cell chamber with multiple design options that can be mixed per experiment. Single reusable carriers and the use of biodegradable plastics, in a hybrid of FDM and MSLA manufacturing methods, reduce plastic waste. The system is easy to adapt to bespoke designs, with concept-to-prototype in a single day, offers significant cost savings to the users over commercial sources, and no loss in dimensional quality or reliability.
Mapping CGRP-IR innervation of male mice stomach with Neurolucida 360 v1
Authors: Nguyen Duyen , Ma Jichao
Published: May 2022

This protocol describes the process of using Neurolucida 360 software to map the topographical organization of Calcitonin gene related peptide – immunoreactive axons and terminals in the muscular layer of mice stomach. Stomachs were removed, layers were separated and gone under immunohistochemistry as whole mounts, then scanned using confocal microscopy.
DRG stimulation - colorectal afferents v1
Authors: not provided longtu.chen , Feng Bin
Published: May 2022

Assessing the efficacy of DRG stimulation to block distension-evoked (mechanical) afferent transmission using the colorectum-PN-DRG-DR preparation.
Prescription patterns of somatostatin analogues in patients with acromegaly and neuroendocrine tumors v1
Authors: Machado Alba Jorge
Published: May 2022

Objective. Acromegaly and neuroendocrine tumors are rare diseases that, under certain conditions, can be treated with somatostatin analogues. The aim was to determine the prescription patterns of somatostatin analogues in a group of patients with acromegaly and neuroendocrine tumors affiliated with the Colombian Health System. Design. A retrospective study. Methods: A cohort of patients from a drug dispensing database that collected all prescriptions of long-acting somatostatin analogues (octreotide, lanreotide, pasireotide). Sociodemographic variables, clinical variables (diagnosis and comorbidities) and pharmacological therapy variables (dose, changes, persistence of use, comedications) were considered. Results: A total of 213 patients were identified, including 139 (65.3%) with acromegaly and 74 (34.7%) with neuroendocrine tumors. There was a predominance of women (58.7%) and a mean age of 59.7 ± 14.5 years. The most commonly used medications were lanreotide autogel (n = 107; 50.2%), octreotide LAR (n = 102; 47.9%) and pasireotide LAR (n = 4; 1.9%). During follow-up, 11.3% of patients experienced modifications of therapy, with a mean duration from the beginning of treatment to the change in medication of 25 ± 15.9 months. A total of 48.9% of the patients with acromegaly and 87.1% of individuals with neuroendocrine tumors received maximum approved doses of the drug. Conclusions: Patients with acromegaly and neuroendocrine tumors in Colombia are mainly women and are most frequently treated with lanreotide autogel for acromegaly and with octreotide LAR for neuroendocrine tumors. In addition, a high proportion are managed with maximum doses of long-acting somatostatin analogues.
Plate Protein Expression on Autoinduction media v3
Authors: Fadanka Stephane , Gandini Chiara
Published: May 2022

The current protocol describes the preparation and use of 2X YT autoinduction medium for recombinant protein expression on Petri dishes. This protocol allows for reproducible and time effective expression experiments to be undertaken with minimal user intervention as compared to standard procedures using IPTG.
SPARC_CWRU_Shoffstall_OT2-OD025340_VagusNerveOsmiumTetroxideStaining V.1 v1
Authors: R. Upadhye Aniruddha , A Pelot Nicole , A. Ludwig Kip , J. Gustafson Kenneth , Shoffstall Andrew
Published: May 2022

In this protocol, we describe our steps to stain nerve fibers with Osmium Tetroxide for further visualization wtih microCT, 3D MUSE (Microscopy with Ultraviolet Surface Excitation), and electron microscopy.
Isolation and identification of potential probiotic bacteria from the soil of Saint Martin's island Bangladesh v1
Authors: Mahbubur Rahman Md. , Indra Paul Sulav
Published: May 2022

Soil bacteria of Saint Martin’s Island modulates gut microbiota and prevents Enterococcus faecalis infection in tilapia (Oreochromis niloticus)
Single cell dissociation of brain organoids v1
Authors: not provided michela.deleidi , José Pérez J. María
Published: May 2022

This protocol details about single cell dissociation of brain organoids.
Panicle Ratio Network: A high-throughput dynamic phenotype recognition model based on ultra-high-definition unmanned aerial vehicle images for rice panicle analysis in fields v1
Authors: Guo Ziyue , Yang Chenghai , Yang Wangnen , Chen Guoxing , Jiang Zhao , Wang Botao , Zhang Jian
Published: May 2022

In order to increase the convenience and efficiency of rice cultivation and breeding, a PR evaluation model based on deep learning and UAV–RGB images was established in this study by focusing on the ETP and HD, which affect rice yield and reflect rice growth. Based on an image regression method that reduces the effects of cross-overlapping due to an excessive density of targets, PRNet used a combination of DenseNet and SPP to extract the features of growing panicles during the rice heading stage.The training results showed that the model performed well with a data set collected in 2019, and the estimation results obtained using the test data set demonstrated that the model had good robustness.Images containing a whole rice plot can be used as direct inputs for PRNet to obtain PR, and then ETP and HD can be determined with high accuracy according to the PR curve.After testing images with different resolutions in the model, the acceptable resolution range for the model was determined to be between 0.6 mm and 2.4 mm when the RMSE for the estimated results was less than 15%. Thus, data collection schemes were presented to facilitate the use of PRNet.However, different types of cameras have different image quality settings, and the model's estimation accuracy might not be ideal even if the resolution is acceptable.The accuracy of the estimations obtained by the model were higher for rice plots in the center of images captured around noon compared with those at other times or at the edges of images.Based on a high-throughput and efficient UAV–ultra-high-definition imaging platform, PRNet is suitable for nondestructive PR monitoring in more than 2,000 plots within 10 min.Furthermore, this scheme can be extended to other panicle-related crop phenotypic analyses and provide a reference for extracting indicators determined by several traits, thereby accelerating the development of in situ field crop phenotypic information extraction.
Zebrafish 2021 Environmental Summary, Reptile & Aquatics, Stowers Institute for Medical Research v1
Authors: P Baumann Diana , Petrie Adam
Published: May 2022

Zebrafish are an established model organism in biomedical research, and we have maintained these fish at the Stowers Institute for Medical Research since 2006. The housing systems and facility rooms allow for standardization of husbandry and environmental parameters. Acknowledging that husbandry conditions and environmental parameters can impact research results we provide information about the housing, nutrition, maintenance, and health for our zebrafish colonies. This information will be applicable to any zebrafish housed in the facility in 2021.
The role of mutuals in social protection policies: innovative experiences around the world v1
Authors: Niang Marietou , Gélinas Émilie , Mallé Samb Oumar , Ridde Valery
Published: May 2022

The objective of this scoping review is to produce a state of knowledge on the experiences of countries that have used community-based or health mutuals in their national social protection systems and the functions delegated to these mutuals. As this topic is to our knowledge not very developed in the literature, our review will be extended to all countries of the world. There are two main questions to be answered: 1.What are the existing experiences in the world that have usedcommunity-based health insurance orhealth mutuals in their national social protection policies? 2.What functions have been delegated to these mutuals in these country experiences?
cDNA Library Preparation for scRNA-seq (10x Genomics) v1
Authors: not provided molmer , Lotz Martin , Mondala Tony , Head Steven
Published: May 2022

Samples are processed using V2 barcoding chemistry kits of 10x Genomics. For each run, 10,000 cells from individual donors arelabeled with distinct oligo-barcoded antibodies (cell hashing), enabling us to pool samples from both conditions on each 10X Genomics run and reduce batch effects.All samples from a given experiment areprocessed in parallel in the same thermal cycler. The 10x user guide can be found https://assets.ctfassets.net/an68im79xiti/1C16trEdzy1Folq5xbOijE/7e6fb1f504e130bd561d898384da99d9/CG000315_ChromiumNextGEMSingleCell3-_GeneExpression_v3.1_DualIndex__RevB.pdf and is also an attached document.
Western blot (NuPAGE and MES buffer) v1
Authors: Smith Laura
Published: May 2022

Cells were lysed in 1% Triton X 100 lysis buffer with protease and phosphatase inhibitors. Cell lysates were electrophoresed with NuPage™ Bis-Tris protein gels. Proteins were transferred to a PVDF membrane, blocked in 10% milk and treated with primary and secondary antibodies in 5% milk. Antibody binding was detected using the GE Healthcare Amersham™ electro-chemi-luminescence (ECL)™ Prime Western Blotting Detection Reagent.
Quantitative real-time PCR of mRNA v1
Authors: Smith Laura
Published: May 2022

Total RNA was extracted from dry cell pellets using the RNAeasy kit (Qiagen), per manufacturer’s instructions. Concentration and purity of ribonucleic acid (RNA) was determined using the Nanodrop. 600 ng RNA was converted to cDNA with the QuantiTect reverse transcription kit (Qiagen) as per manufacturer’s instructions. Quantitative real-time polymerase chain reaction (PCR) reactions were performed using the QuantiTect SYBR Green kit (Qiagen). To summarise, 2 mL cDNA, 10 mL SYBR Green, 7 mL RNAse free dH2O and 1 mL 20 mM forward and reverse primers was added to each well. A mock mixture using 2 mL RNAse free dH2O instead of cDNA was used as a blank. Primers used (Eurofins Genomics). For the PCR reaction activation was at 95°C for 10 minutes. Following this was 40 cycles of denaturing at 94°C for 15 seconds; annealing at 58°C for 30 seconds and extension at 72°C for 30 seconds. Fold-change in gene expression was calculated using the ΔCT method, based on biological reference samples and glyceraldehyde 3-phoshate dehydrogenase (GAPDH) mRNA levels for normalisation. All results obtained were from the evaluation of two technical duplicates of three independent experiments.
Anna's IF Protocol for Ibidi Slides v1
Authors: Bora Akmeriç Emir
Published: May 2022

Immunostaining protocol for ibidi perfusion slides
In Vivo GCase Activity Assay v1
Authors: Smith Laura
Published: May 2022

Assay uses the substrate PFB-FDGluc (5-(Pentafluorobenzoylamino)Fluorescein Di-β-D-Glucopyranoside)from ThermoScientific (Cat# P11947). Substrate is supposed to be taken up by late endosomes and lysosomes only and fluoresces green when cleaved by lysosomal GCase. Described by Mazzulli et al., J Neurosci. 2016 Jul 20;36(29):7693-706.
High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR v4
Authors: Topol (Verily Life Sciences) Aaron , not provided marlene.wolfe , White (Verily Life Sciences) Brad , Wigginton Krista , B B Boehm Alexandria
Published: May 2022

v3 updates: changes to the S gene primers and probes for detection of all SARS-CoV-2 variants This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and ORF1a and a duplex assay targeting Bovine Coronavirus Vaccine (BCoV) and Pepper Mild mottle virus (PMMoV) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. This protocol describes 2 separate PCR reactions, one containing primer/probe mixtures targeting the three SARS-CoV-2 targets and one containing primer/probe mixtures targeting BCoV and PMMoV. BCoV is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition. PMMoV is an enveloped virus which is abundant in human fecal waste and serves as an endogenous control for data normalization. PMMoV RNA is abundant at such high levels in wastewater samples that the samples must be diluted by a factor of 100 before quantification. The readout of this assay is a concentration of each target in the extracted RNA samples (copies/uL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.
Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y,del156-157/R158G, del143-145, and LPPA24S) in settled solids using digital RT-PCR v5
Authors: Hughes Bridgette , J. White Bradley , K. Wolfe Marlene , B B Boehm Alexandria
Published: May 2022

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one each for HV69-70, E484K/N501Y, del156-157/R158G, del143-145, and LPPA24S) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.
Optimizing the electroporation parameters forHeterometopus palaeformis (strain RAJCA) v1
Authors: Gomaa Fatma , Rotterova Johana , Beinart Roxanne , Pachiadaki Maria , Edgcomb Virginia
Published: May 2022

Cell viability after incubation with different electroporation buffers, Phosphate buffered saline (PBS) is used as an electroporation buffer for many eukaryotic cells. However, we observed that the PBS has a detrimental effect on the cell, causing disruption in H. palaeformis cell membrane. We then tested the tolerance of H. palaeformis to three different electroporation buffers: A-Gene Pulser Electroporation Buffer(BIO-RAD, USA) B-Iso-osmolar Buffer (Eppendorf, USA) C-Hypo-osmolar Buffer (Eppendorf, USA) Cells were incubated in each buffer independently for 10 minutes and observed for their viability under inverted microscopy. For all the three tested buffers, we noticed that once buffer was added, the cells showed instant osmic shock, include loss of motility and relative shrinking in the cell membrane (Fig. 1). After 10 minutes incubation period in each buffer, cells were transferred to their Cerophyl culture medium to recover. We observed progressive recovery of the cells. Table 1 shows the time that took the cells to completely recovered and the percentages of the recovered cells after incubation at the three different electroporation buffers. Table 1 Electroporation buffer Duration of recovery Percentage of the recovered cells Gene Pulser 10 minutes >90% Iso-osmolar 10 minutes 70% Hypo-osmolar 2-5 minutes 70% Fig.1. Light microscopy images showing the effect of the different electroporation buffer on the H. palaeformis cell membrane; PBS has detrimental effect on the cell causing disruption of cell membrane.
Passaging of cells v1
Authors: Krähenbühl Guido
Published: May 2022

General Lab procedure, Cell passage
Region velocity estimation and visulization with seurat v1
Authors: Zhang Chen
Published: May 2022

Example pipeline of region velocity estimation and visulization of steady-state model and dynamical model using EM algorithm in R with Seurat to pretreat scRNA-seq data
Quantification of SARS-CoV-2 variant mutations (HV69-70, E484K/N501Y,del156-157/R158G, del143-145, and LPPA24S) in settled solids using digital RT-PCR v6
Authors: Hughes Bridgette , J. White Bradley , K. Wolfe Marlene , B B Boehm Alexandria
Published: May 2022

This process instruction describes the steps for quantitative analysis of nucleic acid from SARS-CoV-2 with a triplex Reverse Transcriptase droplet digital Polymerase Chain Reaction (RT-ddPCR) assay targeting the N Gene, S Gene and 3 mutation assays (one each for HV69-70, E484K/N501Y, del156-157/R158G, del143-145, and LPPA24S) in extracted and purified RNA samples from solid wastewater samples for population level SARS-CoV-2 community surveillance. RT-ddPCR is a modified version of conventional RT-PCR workflows which involves separating the reaction mixture into many partitions (~20,000) before thermal cycling which allows for direct absolute quantification of the target RNA molecules. Future protocols will be published that are complementary to this one and describe assays targeting additional SARS-CoV-2 mutations. This protocol uses RNA extracted using this protocol: High Throughput RNA Extraction and PCR Inhibitor Removal of Settled Solids for Wastewater Surveillance of SARS-CoV-2 RNA. That RNA is generated from samples subjected to pre-analytical steps outlined in: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses. It is recommended that these assays be run along assays for PMMoV and BCoV as controls as described in the companion protocol High Throughput SARS-COV-2, PMMOV, and BCoV quantification in settled solids using digital RT-PCR The readout of this assay is a concentration of each target in the extracted RNA samples (copies/µL). Scope This process instruction applies to quantitative analysis of nucleic acid from SARS-CoV-2 RNA from solid wastewater samples with ddPCR using a Bio-Rad AutoDG Droplet Digital PCR system consisting of the AutoDG Automated Droplet Generator and the QX200 droplet reader.
JAX - EZ Lysis Nuclei Isolation for 10x Genomics Assays v3
Authors: Bessonett Shannon , A Perry Greggory , L Daigle Sandra , not provided Paul.gabriel , Luo Diane , Grassmann Jessica , F F Flynn William , Courtois Elise , Robson Paul
Published: May 2022

The purpose of this protocol is to produce single nuclei from frozen human tissues for downstream assaying with the 10x Genomics Multiome assay. This protocol has been demonstrated using Human placenta tissue as well as human Glioblastoma. This protocol is modified from Sigma Aldrich Nuclei Isolation Kit: EZ Prep protocol and 10x Chromium Demonstrated protocol for Single Cell Multiome ATAC + Gene Expression Sequencing (CG000365).
Electroporation of fluorescent antisense molecules into Bodo saltans and live imaging v1
Authors: Mastaneh Ahrar not provided , not provided g.hurst , Chrostek Ewa
Published: May 2022

This is the protocol used in our Laboratory at the University of Liverpool to electroporate molecules into Bodo saltans.
Protein extraction, quantification, and western blot for Bodo saltans v1
Authors: Chrostek Ewa , Mastaneh Ahrar not provided , Gregory Dd Hurst not provided
Published: May 2022

This protocol is used in our Laboratory in Liverpool to work with proteins from Bodo saltans and other kinetoplastids (eg. trypanosomes).
[RETRACTED] Titan XL Male Enhancement : Titan XL Testosterone Enhancer Work Reviews 2022 v1
Authors: not provided stashkevich
Published: May 2022

[RETRACTED]

Titan XL Male Enhancement:is a penis enhancement formula that claims to change your life.By taking two capsules of Titan XL Male Enhancement daily,
Team Deleidi | Protocols | "Glucocerebrosidase, a Parkinson´s disease-associated protein, is imported into mitochondria and regulates complex I assembly and function" v1
Authors: not provided michela.deleidi , José Pérez J. María , Raji Hariam , Baden Pascale , Bertoli Federico
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"
Custom Peptide Design v1
Authors: Miller Cathy
Published: May 2022

Interested in the use of biologically active proteins and peptides as potential therapeutic agents has grown dramatically in recent years. Although many meaningful studies can be performed, it is important to consider several factors such as the length of sequence, amino acid residues and the sequence of peptides, all of which will influence whether correct assembly and purification are feasible. If you would like Creative Peptides to help you in designing your peptide, our technical support department can assist you.
[RETRACTED] Guardian Botanicals Blood Balance Australia 2022: Does It Work? My Latest Report v1
Authors: not provided health
Published: May 2022

[RETRACTED]

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PNA Synthesis v1
Authors: Miller Cathy
Published: May 2022

Peptide nucleic acid is one of the more important oligonucleotide mimics. Although PNA has a significant change in structure relative to oligonucleotides, the binding between PNA and complementary nucleic acids still follows the principle of base complementary pairing, it even has higher affinity than natural nucleotides, and has stronger resistance to degradation by nucleases and proteolytic enzymes. Researchers have designed and synthesized PNA with various structures to optimize its biological stability and availability, target binding characteristics, and pharmacokinetic characteristics. We provide customers with various types of PNA conjugates and tailor-made PNA products for customer projects to achieve scientific research purposes. Our product analysis service will help our customers perform product analysis without worrying about poor product quality.
PNA/DNA Chimera Synthesis v1
Authors: Miller Cathy
Published: May 2022

Pure PNA has significant binding properties, but it has completely different properties from DNA and RNA. For example, it cannot be recognized as a substrate when it interacts with nucleic acid modifying enzymes. On the one hand, this is an advantage, because it makes PNA very stable for the degradation of nucleases; but on the other hand, it also limits certain applications of PNA, such as not being used as a substrate for DNA polymerase, DNA kinase or DNA ligase. In one molecule, the combination of PNA and DNA can produce PNA/DNA chimeras with new properties. This substance significantly improves the solubility of PNA in aqueous solutions, PNA/DNA chimera can be analyzed and purified by PAGE and ion exchange chromatography because the DNA part is negatively charged. Interestingly, PNA/DNA chimeras also have biological functions, such as being used as primers for DNA polymerases or binding to RNA to mediate the dissociation of RNase H. Finally, the study of PNA/DNA chimeras can provide useful information about the structural and functional properties of DNA and RNA themselves. Creative Peptides provides PNA/DNA chimera related services, we can provide design and synthesis integrated services, and committed to helping you solve synthesis problems and customize high-stability, multi-type PNA/DNA chimera products.
Total Peptide Library Construction Technology v1
Authors: Miller Cathy
Published: May 2022

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] Guardian Blood Balance Australia v1
Authors: not provided health
Published: May 2022

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Peptide Drug Screening v1
Authors: Miller Cathy
Published: May 2022

Peptides are ideally suited to mimic natural ligands and usually act in an antagonistic or agonistic manner. Small active peptide molecules have huge potential to be developed as vaccines, diagnostic reagentsand lead compounds. Therefore, peptides have become the highlight of drug discovery, especially due to their small size, structural diversity and high specificity for a given target. Moreover, peptides can be selected from large natural or artificial peptide libraries rapidly, and are easy for chemical processing and synthesis. Application Peptides are capable of physiologically destroying functional complexes due to their small size and specific binding properties. Peptides perform a variety of biological functions in the body, such as metabolism, digestion, respiration, reproduction, immune defense and many other functions by acting as ligands (such as hormones, neurotransmitters, cytokines, and chemokines). Our Peptide Drug Screening Services Creative Peptides provides a wide range of screening strategies to meet the needs of every customer for peptide drug screening on time and budget. Our skilled and dedicated scientific researchers have rich experiences in selecting the most suitable methods and techniques for each peptide drug project. Over the years, the excellent quality and reliability of our peptide services have been appreciated by customers worldwide.
Linker Design and Optimization v1
Authors: not provided boc.protac
Published: May 2022

The linker is responsible for linking two key domains of PROTAC: the domain that specifically binds to the target protein that needs to be degraded, and the domain that binds to the E3 ubiquitin ligase. To date, several types of connectors have been reported and used in the formation of PROTAC ternary complexes, such as PEG liner, alkyl liner and "click chemical" liner. It plays an important role in the effective ubiquitination and final degradation of target proteins. As a leading service provider in drug discovery and research, BOC Sciences is fully qualified and committed to providing one-stop PROTAC®development, which has become a promising strategy in the field of small molecular drug discovery. With a comprehensive and advanced platform, we provide Linker Design and Optimization Services to customers around the world to meet new drug discovery goals.
PROTAC diastereomer Design（negative control） v1
Authors: not provided boc.protac
Published: May 2022

Diastereomers are stereoisomers in which molecules have two or more chiral centers and the relationship between molecules is not mirrored. In PROTAC drug development, in order to evaluate the pharmacokinetics of a single diastereomer or mixture of diastereomers, manufacturers should quantitatively analyze samples from the early stages of drug development for a single diastereomer in vivo. This will allow the assessment of the possibility of mutual transformation as well as the distribution of absorption, distribution, biotransformation and excretion (ADBE) of individual isomers. When the drug is a raceme and the pharmacokinetic characteristics of the isomers are different, the manufacturer should monitor the diastereomer separately to determine properties such as dose linearity and the effects of metabolic or excretory changes and drug-drug interactions. As a leading service provider in drug discovery and development, BOC Sciences is fully qualified and committed to providing one-stop PROTAC®development services, which has become a promising strategy in the field of small molecular drug discovery. With a comprehensive and advanced platform, we design PROTAC diastereomer to customers around the world to meet new drug discovery goals.
[RETRACTED] Power Keto Gummies - Lose Weight Faster! | Special Offer! v1
Authors: not provided jeneferloma
Published: May 2022

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Power Keto Gummies
PROTAC GMP Services v1
Authors: not provided boc.protac
Published: May 2022

BOC Sciences’ GMP production sites were designed in strict accordance to US-FDA guidelines for GMP Manufacturing. It has passed US-FDA inspections. We provide standard QC tests before releasing GMP products. For GMP products, we guarantee >98% purity. In addition, extra tests can be provided according to the requests of clients. Our GMP manufacturing capabilities are designed to meet actual and future customer needs in respect of all stages of PROTAC drug discovery. Advantages Strategic variety encompassed in the combinations of linkers and ligands aids the design of target degraders State-of-the-art techniques and software tools for ligand design and screening Standard QC tests before releasing GMP products Industry-leading turnaround times on deliveries worldwide-and highly competitive pricing Numerous purification options, such as liquid chromatography isolation, silica gel purification, and high-performance liquid chromatography Both research-grade or GMP-grade ligand and linker can be customized. cGMP Manufacturing that is fully traceable
[RETRACTED] Power Keto Gummies Reviews! v1
Authors: Keto Gummies Reviews Power
Published: May 2022

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High Efficiency Transformation Protocol using NEB 10-beta Competent E. coli (C3019) v6
Authors: England Biolabs New
Published: May 2022

This is the High Efficiency Transformation Protocol for C3019H and C3019I cells.
NEBNext® Ultra™ DNA Library Prep Protocol for Illumina® (E7370) v3
Authors: England Biolabs New
Published: May 2022

The NEBNext®Ultra™ DNA Library Prep Kit for Illumina®contains reagents for preparation of libraries for next-generation sequencing on the Illumina platform from 5 ng – 1 µg input DNA, in a streamlined workflow. Please note that adaptors and primers are not included in the kit and are available separately. Each kit component must pass rigorous quality control standards, and each set of reagents is functionally validated together by construction and sequencing of a library on the Illumina sequencing platform. For larger volume requirements, customized and bulk packaging is available by purchasing through the OEM/Bulks department at NEB. Please contact custom@neb.com for further information.
Suggested protocol for loading a DNA Ladder/marker v2
Authors: England Biolabs New
Published: May 2022

This is the suggested protocol for use with: Quick-Load®Purple 1 kb DNA Ladder (N0552) Quick-Load®Purple 100 bp DNA Ladder (N0551) Quick-Load®Purple 50 bp DNA Ladder (N0556) Quick-Load®1 kb Extend DNA Ladder (N3239) Quick-Load®1 kb DNA Ladder (N0468) Supercoiled DNA Ladder (N0472S) λ DNA-Mono Cut Mix (N3019) фX174 DNA-HaeIII Digest (N3026) pBR322 DNA-BstNI Digest (N3031) pBR322 DNA-MspI Digest (N3032) 2-Log DNA Ladder (0.1-10.0 kb) (N3200) 100 bp DNA Ladder (N3231) 1 kb DNA Ladder (N3232) Low Molecular Weight DNA Ladder (N3233) 50 bp DNA Ladder (N3236)
Masson's Trichrome Staining Protocol for FFPE spheroids and microtissues v1
Authors: Denn Angela , Mikulski Zbigniew
Published: May 2022

The following protocol has been optimized for image analysis-based quantification of collagen within FFPE spheroid samples. It is run on a Leica ST5020 automated stainer. This protocol can be adapted to manual staining. PURPOSE: Trichrome stains are used to differentiate collagen from other tissues. They are usually nuclear and cytoplasmic stains, followed by differentiation, then followed by a collagen fiber stain. Trichrome stains are useful for indicating fibrotic changes. PRINCIPLE: The staining reaction is based on the differential effect of acid dye on collagen and other tissue components.
Non-radioactive phosphorylation with T4 PNK 3' phosphatase minus (M0236) v2
Authors: England Biolabs New
Published: May 2022

Protocol for non-radioactive phosphorylation with T4 PNK 3' phosphatase minus.
[RETRACTED] Apple Keto Gummies Australia v1
Authors: not provided soufianeelbekri
Published: May 2022

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Quite possibly the most well known ways for individual to get in shape and cut back excess is the keto diet. Today, we will enlighten you concerning Apple Keto Gummies Australia diet pills. This new
Shipping protocols v1
Authors: J J. Barrero Gabriel , Palmer Abraham , Polesskaya Oksana
Published: May 2022

Shipping tissue protocol