eamag/protocols-with-steps huggingface dataset:
TITLE: FAST.R Installation and Launch with RStudio
AUTHORS: Francesco Neri

[DESCRIPTION]
Step -by-step protocol to setup the R shiny application FAST.R for data analysis and visualization of FAST-generated data. This protocol is meant to be used within the FAST workflow

[STEPS]
SECTION: Install FAST.R with RStudio
1. If familiar with R and RStudio, see the FAST.R package page for installation and use.

If NOT familiar with R and RStudio, read instructions below
SECTION: Install FAST.R with RStudio
3. Install FAST-R

 

SECTION: Install FAST.R with RStudio
2.1. Download and install R and RStudio
Download from here. Note that R version 4.3.2 or higher is required.
SECTION: Install FAST.R with RStudio
2.2. Download the files below by clicking on each link; then click the "Download raw file" button
Install FASTR
Run FASTR

 

 

SECTION: Install FAST.R with RStudio
3.1. If you get the message "These packages have more recent versions available", choose update all
click in the Console > type "1" > press Enter

 

SECTION: Install FAST.R with RStudio
4. Launch FAST-R

 

SECTION: Install FAST.R with RStudio
4.1. Once FAST-R opens, simply follow the instructions provided directly in the app to perform data analysis and/or generate graphs
SECTION: Install FAST.R with RStudio
2. If you have never used FAST.R via RStudio before, follow Steps 2-3. Otherwise, skip to Step 4

FAST.R setup
TITLE: RNA Isolation for Tissue using TRIzol
AUTHORS: Sze-Xian Lim

[STEPS]
?. Add of TRIzol per of tissue and homogenize using handheld homogenizer.
[TRIzol]
[tissue]
?. Incubate at  for to allow nucleoprotein complexes to dissociate.
?. Centrifuge at max speed for at .
?. Carefully remove the top aqueous phase and transfer to a new Eppendorf tube. The interphase and bottom organic phase can be saved for DNA and protein respectively.
?. Add  of 100% isopropanol to the aqueous phase, mix by inversion and incubate at for a minimum of .
[100% isopropanol]
?. Spin down at maximum speed for 30 mins to precipitate RNA.
?. Remove supernatant, and add of ethanol to wash pellet.
1 ml
[ethanol]
?. Add of chloroform to each ml of TRIzol carefully and vortex to mix well.
[chloroform]
?. Spin down at max speed for 15 minutes and remove supernatant.
?. Resuspend pellet in appropriate volume of nuclease free water.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol of Electroacupuncture (EA) treatment on sub-acutely aging rats.</p>

[BEFORE_START]
<p>Sterile acupuncture needles [size:φ0.30*25 mm (diameter:0.30 millimeter, length: 25 millimeter); made by Suzhou Acupuncture ＆ Moxibustion Appliance Co., Lid, Suzhou, P.R. China] and the pulse generator (6805-II, Shanghai Taicheng technology development co ., LTD) were used during the EA treatment.</p>

[STEPS]
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TITLE: Publication Trends in Drug Delivery and Magnetic Nanoparticles
AUTHORS: Saba Ale Ebrahim, Amirhossein Ashtari, Maysam Zamani Pedram, Nader Ale Ebrahim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This bibliometric study investigated the public trends in the fields of nanoparticles which is limited to drug delivery and magnetic nanoparticles’ literature published from 1980 to October 2017. The data were collected from the Web of Science Core Collections, and a network analysis of research outputs was carried out to analyse the research trends in the nanoparticles literature. Nanoparticles and its applications are progressing in recent years. The results show that documents in the field of nanoparticles in chemistry and material science have improved in citation rate, as the authors were researching in multidisciplinary zones. Top-cited documents are mainly focusing on drug delivery, magnetic nanoparticles and iron oxide nanoparticles which are also the top research keywords in all papers published. Top-cited papers are mostly published in Biomaterials journal which so far has published 12% of top-cited articles. Although research areas such as contrast agents, quantum dots, and nanocrystals are not considered as the top-ranked keywords in all documents, these keywords received noticeable citations. The trends of publications on drug delivery and magnetic nanoparticles give a general view on future research and identify potential opportunities and challenges.</div></div>

[STEPS]
TITLE: Illumina (post-MR DNA) processing pipeline : mothur
AUTHORS: Kylie Langlois

[DESCRIPTION]
<p>This pipeline is for datasets generated from Illumina (2x300) sequencing by MR DNA. MR DNA runs sequences through a proprietary pipeline for quality control of sequences and OTU clustering. This pipeline begins with the OTU abundnace table and utilizes the representative sequence set generated by the MR DNA pipeline. <strong>Through trial and error, I have discovered the easiest and best practice for my lab is to use a combination of QIIME (full installation) and mothur. This protocol is the mothur portion.</strong> This protocol does not include data visualization.</p>
<p> </p>
<p>As of 4/5/17, the following versions were used: QIIME v.1.9.1-amd64.vdi and mothur v.1.39.5</p>

[STEPS]
?. [Before you start this protocol]
Begin with Illumina (post-MR DNA) processing pipeline : QIIME steps 1-7 to rarefy the OTU abundance table. Complete step 12 to convert the rarefied OTU abundance table to a text file.
?. [Assign taxonomy]
To compare the taxonomy assigned by the sequencing facility (MR DNA in our case) through a proprietary pipeline and curated database, you can use the classify.seqs command in mothur.
?. [Install the executable version of mothur in Windows]
"Downloading an executable version of mothur is the easiest and fastest way to get started." -mothur download pageThe best way to use mothur is in Windows or Mac where you can download an execuatable version of mothur that runs through a terminal window. All scripts are written in C++, a wrapped language, so you can execute a command within the mothur terminal just like you would execute a QIIME command in the QIIME terminal window. Here is the latest release of mothur on github. Follow instructions of the installation page. If you download the executable version of mothur, it will simply show up as an icon that opens to a terminal window when double-clicked. That is mothur! It is simpler than it appears.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>With the Fall Creators update to <strong>Windows 10</strong> comes the ability for all to install <strong>Ubuntu</strong> on your Windows 10 machine by simply downloading the app from the official <a href="https://www.microsoft.com/en-gb/store/p/ubuntu/9nblggh4msv6" target="_blank" rel="noopener noreferrer"><strong>Windows App Store</strong></a>. Rather than using a tool like <strong>Cygwin</strong>, which replicates a <strong>UNIX(-like)</strong>experience with some of the same command line tools, <strong>Ubuntu on Windows</strong> is the full <strong>Linux </strong>experience. Unlike using Cygwin, you can install bioinformatics software like <strong>Salmon</strong> or <strong>Cufflinks</strong> with ease. This protocol from <a href="https://www.badgrammargoodsyntax.com/" target="_blank" rel="noopener noreferrer">Bad Grammar, Good Syntax</a> provides a detailed method of how to install Ubuntu, setting up Python, and installing binaries of software for compbio. </p>

[BEFORE_START]
<p>You need a computer with Windows 10 installed and kept up to date. </p>

[GUIDELINES]
<div>
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<div>
<p>In the past, you could try to dual boot your system so that it has two operating systems or run Ubuntu in a virtual machine within Windows, but I think this method is nice and clean. Others will disagree. </p>
<p> </p>
<p>What you won't get is the Ubuntu graphical user interface. You are still stuck with running Windows 10. If that is a problem for you, then dual booting or a virtual machine is probably your best option. Instead, what Ubuntu on Windows offers is a simple way to get access to the command line from Ubuntu. Not only does that mean that you can run software designed and compiled to run on Linux, but you can also use all the cool tools of Ubuntu like apt-get to seamlessly install programs, keep them up-to-date, and fetch dependences. </p>
<p> </p>
<p>An alternative guide to Linux on Windows using Conda:<br /><a href="https://github.com/kapsakcj/win10-linux-conda-how-to/blob/master/README.md" target="_blank" rel="noopener noreferrer">https://github.com/kapsakcj/win10-linux-conda-how-to/blob/master/README.md</a></p>
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[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>

[STEPS]
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TITLE: LAMP Master Mix + Reaction Protocol
AUTHORS: Caterina Villari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the LAMP (Loop-mediated isothermal amplification) master mix and reaction.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Elevated plus maze protocol
AUTHORS: Archana Venkataraman, Holly Ingraham

[DESCRIPTION]
This protocol describes steps for assessing anxiety-like behavior in rodents using an elevated plus maze test. 

The elevated plus maze (EPM) is one of the widely used behavioral assays to measure anxiety-like behaviors in rodents.  In the EPM test, rats or mice are placed at the intersection of a plus-shaped maze with two open arms and two closed arms. The rodents show natural preference to stay in closed areas and avoid open and elevated areas. This expression of unconditioned anxiety is well captured in this assay.The amount of time spent in the open arm vs the closed arm is typically measured to assess anxiety-like behavior. The anxiolytic or anxiogenic effects of pharmacological agents and genetic manipulations can be investigated using this method.

[BEFORE_START]
Pre-handling the mice by the experimenter at least for 3-5 days prior to testing is strongly suggested. 


[GUIDELINES]
Male and female mice should be tested separately on different days to avoid the effect of pheromones on behavioral output. 
It is suggested that the researchers performing the behavioral assays be blinded for the mouse genotype to remove experimenter bias.

[STEPS]
1. Make sure the equipment is clean and dry before use. 
 

12. The time spent and distance traveled in the open and closed arms were determined using ANY-maze software and these measures serve as an index of anxiety-like behavior.
 

6.  Once ready to start the experiment, place a mouse in the center area of the maze.
2. Set up the overhead camera and ensure that the open arms and closed arms are appropriately selected in the 'zone' tab of the ANYmaze program. 
3. Enter all animal information such as animal ID, status, and treatment, in the ANYMaze software. 
4. Allow experimental animals to habituate to the behavior testing room at least 45 min prior to beginning of the test.
9. Allow the animal to explore undisturbed for 5 min. 
11. After each trial, all arms and the center area were cleaned with 70% ethanol, to remove olfactory cues. 
8. Immediately after the animal is placed in the maze, start the experiment on the ANYmaze software.
10. Each animal is given one trial and should not be subjected to repeated testing. 
7. Place the animal such that the head is oriented towards the closed arm and away from the experimenter. Always handle each animal consistently throughout all experiments. 
5. Drug administration: In experiments involving chemogenetic manipulation, deschloroclozapine (DCZ) injections (75 µg/kg) were administered intra-peritoneally 10 mins before testing. For alosetron pre-treatment, mice were subcutaneously injected with 0.1 mg/kg of the 5HT3 antagonist 15 mins prior to the DCZ injections.
TITLE: Reference Kidney Sample Collection
AUTHORS: Nicholas Lucarelli

[DESCRIPTION]
Inclusion:
Minimal pathologic changes

Exclusion:

Evidence of hydronephrosis
Evidence of infectious disease
Proteinuria
Image artifacts
>5% Interstitial Fibrosis and Tubular Atrophy (IFTA)

[STEPS]
SECTION: Sample Collection
1. Patient samples will be obtained from human kidneys, using nephrectomies from renal cancer patients. Tissue sections will be uninvolved and away from tumor areas. Sections will be screened by a pathologist to ensure minimal pathologic changes, and minimal image artifacts.
SECTION: Sample Collection
2. Inclusion/Exclusion Criteria:

Inclusion:
Minimal pathologic changes

Exclusion:

Evidence of hydronephrosis
Evidence of infectious disease
Proteinuria
Image artifacts
>5% Interstitial Fibrosis and Tubular Atrophy (IFTA)
TITLE: Analysis of the reproducibility, transparency and quality of economic studies on pharmacological interventions in health included in clinical practice guidelines in Spain 2000-2020
AUTHORS: juanruanoruiz  ,   Pedro J Gómez Arias,   Jesús Gay-Mimbrera,   Macarena Aguilar-Luque,   Beatriz Isla-Tejera

[DESCRIPTION]
Hypothesis
The degree of transparency and reproducibility of economic health analyses, whether primary studies or systematic reviews and meta-analysis, which are used for development could condition the usefulness of clinical practice guidelines in which they are included regardless of the methodological quality of them. 

Objectives
Primary objective
Analyze the relationship between the methodological quality of clinical practice guides published in Spain and the current state of reproducibility and transparency of the health economic assessment studies included in them, both primary studies or systematic reviews and meta-analysis. 

Secondary objectives 
Evaluate the quality of CFCs produced in Spain in the period 2000-2020. 
Evaluate the methodological quality of the economic assessment studies included in the GPCs produced in Spain in the period 2000-2020. 
Describe the transparency and reproducibility of the selected economic assessment studies. 
Analyse whether there are differences in transparency and reproducibility of economic assessment studies depending on the quality of the GPCs that include them. 
Analyse whether there have been improvements in the transparency and reproducibility of economic assessment studies by comparing the GPCs containing them that have been produced in 2000-2012 vs 2013-2020.

[STEPS]
TITLE: Human primary T cell culture media
AUTHORS: Bulent Arman Aksoy, Pınar Aksoy, Megan Wyatt, Chrystal M. Paulos, Jeff Hammerbacher

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. [Mix components]
Mix all in a 500 ml 0.22 um filter bottle in the hood:
?. [Mix components]
500 ml RPMI-1640 (Corning™ cellgro™ RPMI 1640 Medium (Mod.) 1X with L-Glutamine)
?. [Mix components]
25 ul of EMD Millipore™ Calbiochem™ β-Mercaptoethanol, Molecular Biology Grade (715 uM)
?. [Mix components]
12.5 ml of HyClone™ HEPES Solution (25 mM)
?. [Mix components]
50 ml of FBS (Fetal Plus®)
?. [Mix components]
5 ml of Penicillin-Streptomycin (1%)
?. [Mix components]
5 ml of HyClone™ 100mM Sodium Pyruvate Solution (1X)5 ml of HyClone™ Non Essential Amino Acids NEAA 100x solution (1X)
?. [Filter and aliquot]
Sterile filter (0.22um) and aliquot in 50 ml falcon tubes. Keep the media in the fridge, protect from light.
TITLE:      ELISA for quantification of IL-24 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>Monocytes, T and B cells mostly make IL-24. It causes cancer-specific cell death, causes wound healing and protects against bacterial infections and cardiovascular diseases. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-24 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C.
?. Add 50 µl of human serum. Human IL-24 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-24 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-24 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-24 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-24 standard dilutions and the human IL-24 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Rotarod-Test for Mice
AUTHORS: Cristian González-Cabrera, Katharina Draggendorf, Matthias Prigge

[DESCRIPTION]
The Rotarod Test Protocol for Mice assesses their motor coordination and balance. Mice are trained to walk on a rotating rod in a dimly lit environment and then tested for their ability to maintain balance as the rod accelerates from 4 to 40 RPM. The test, repeated thrice with intervals, measures the latency to fall and speed at the fall. This protocol is vital to study neurological disease such as Parkinson disease.

[STEPS]
SECTION: Objective
1. To assess motor coordination and balance in mice
SECTION: Apparatus
2. A suitable Rotarod system for mice, including a rotating rod with separate lanes and fall sensors (e.g., SD Instruments or Med Associates).
Dim red lighting for the test environment - not more the 50lux
SECTION: Preparation
3. Acclimate mice to the test environment and handler before testing.
Bring mice in their home cage to the behavioral room at least 1 hour before testing to minimize stress.
SECTION: Training
4. The goal is for mice to be able to walk forward on the rotating rod.
Place mice in separate lanes on the rod rotating at 5 RPM, allowing them to walk forward to maintain balance.
After 60 seconds on the rod, return mice to their home cage.
Clean the Rotarod with diluted ethanol between training rounds.
Perform a total of three repetitions with a 5-minute interval.
If a mouse falls off before completing 60 seconds, the round can be repeated, but total rounds should not exceed four.
SECTION: Test Procedure
5. Place mice in separate lanes on a rod starting at 4 RPM.
Set the Rotarod to accelerate from 4 to 40 RPM over 300 seconds.
The trial begins with the start of acceleration and ends when a mouse falls off the rod.
If a mouse clings to the rod and completes a full passive rotation, the timer for that mouse is stopped.
Return any fallen mice to their home cage, ensuring minimal disturbance to others still in the trial.
A repeat trial is conducted if the mouse passively rotates or falls off within 5 seconds of the trial start.
Clean the apparatus with Virkon between trials.
Repeat the procedure for a total of three trials with a 10-minute interval between trials.
SECTION: Data Analysis
6. Record and analyze the following parameters
Latency to fall.
Speed at the time of fall.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/CviJI-Purification-From-IL-3A-Virus-Infected-NC64A-er3bd8n" target="_blank">CviJI Purification From IL-3A Virus Infected NC64A Chlorella</a>.

[STEPS]
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TITLE: 709.1 Staining of Dissociated Lung Cells for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE sequencing)
AUTHORS: Gautam Bandyopadhyay, Jeffrey Malik, Heidie Huyck, Ravi Misra, John Ashton, Gloria S Pryhuber

[DESCRIPTION]
This article describes step by step protocol for antibody-staining frozen lung cells for CITE sequencing.

[STEPS]
SECTION: Thawing of frozen dissociated human lung cells
1. Frozen cells were thawed in 37°C water bath and soon after the freezing medium melted cells were transferred into a 15 ml conical tube and the tubes were filled with staining buffer [1% BSA (w/v, Millipore) in DPBS (Biowhittaker)] to dilute the DMSO in freezing medium.
SECTION: Thawing of frozen dissociated human lung cells
2. Cells were centrifuged at 800g, 4°C for 10 minutes.
SECTION: Thawing of frozen dissociated human lung cells
3. After centrifugation supernatants were discarded and cells were resuspended in 1.0 ml staining buffer.
SECTION: Fc receptor blocking and  antibody staining
4. Viable cell counts were determined by trypan blue exclusion assay
SECTION: Fc receptor blocking and  antibody staining
5. Cells were then incubated in staining buffer containing 2% v/v normal mouse serum (Millipore Sigma) for 10 minutes over ice for Fc-receptor blocking.
SECTION: Fc receptor blocking and  antibody staining
6. Cells were then washed with staining buffer and incubated at a final concentration of 1 × 106 cells per 100 μl of staining cocktail containing analyte-specific antibodies (attached table) for 30 min at 4°C.  

SECTION: Fc receptor blocking and  antibody staining
7. After antibody-staining, cells were washed three times (800g, 4°C for 10 minutes) and resuspended in 100 μl staining buffer and passed through a 100-μm strainer (Falcon, Corning) and sent Genomic Research Center, University of Rochester Medical Center for further processing for CITE sequencing.