eamag/protocols-with-steps huggingface dataset:
TITLE: Viruses identification in metagenomes 
AUTHORS: Remi Denise

[DESCRIPTION]
This protocol shows how to use this virus identification workflow (doi: 10.5281/zenodo.7778392) and some manual curation criteria for viral sequence identification in metagenomes.

[BEFORE_START]
This tutorial requires Unix OS with "conda" installed. If you cannot access any Unix OS, you can use virtual machines such as VirtualBox or Vagrant in Windows. If you do not "conda" installed, you can follow the instructions here.

[STEPS]
SECTION: Install dependencies and prep test data
1. Install dependencies

We need the following three tools for this SOP:
snakemake (version >= 7.25.0)
snakedeploy (version >= 0.8.6)

First lets install mamba:

 

Second lets create new conda environment using mamba for this tutorial:

 

Note: When you install the environment you will only need to activate the environment to run the workflow no need to install it again
SECTION: Install dependencies and prep test data
2. Deploy the workflow

To deploy the workflow you need first to activate the environment

 

Then you will need to deploy the workflow depending on the release you want

 

SECTION: Install dependencies and prep test data
3. Preparation of the config file

Now that the workflow is deployed. In the folder where you created, there is a file named config/config.yaml

The important things to change in the file is:

 

If you want the workflow to start after the read assembly you need to indicate the folder of your assemble contigs

 

Note: If you want to start at the assembly steps, just let the value empty with a ""

For the databases for the virus identification only ICTV, refseq viral, IMG/VR and crassphage are mandatory. If you want to add more database add your database to the list in this format

 

Note: everything in the config file could be change and if you miss a value for some mandatory item, the workflow will raise an error
SECTION: Run the workflow
5. Run snakemake workflow

Now everything is configure, you just have to run:

 

The workflow will do all the steps for you and install all the needed software.
SECTION: Run the workflow
4. Activate the snakemake environment

Before running the workflow make sure that the conda environment is activated

 

And that you are in the folder where the workflow is deployed
SECTION: Results
6. When the workflow is done running. You will have a output folder looking like that
TITLE: Generating Non-English IATs and Collecting Offline Samples
AUTHORS: João Oliveira Santos, Emerson Araújo Do Bú, Tomohiro Hara, Cristina Mendonça, Sara Hagá, Ruth Pogacar, Michal Kouril

[DESCRIPTION]
This protocol describes tools to: a) create non-English IATs using iatgen and R, b) build the repository of translations for iatgen, and c) deploy Qualtrics-based IATs in offline environments. Together, these functions increase the flexibility of the IAT and may help reduce the negative effects of WEIRD samples

[STEPS]
TITLE: UC Davis - Maximum passive stiffness (MPS)
AUTHORS: John  Rutledge

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">One index of arterial function that is compromised to a varying degree in individuals with cardiovascular disease is vascular stiffness. This protocol can be utilized to analyze vascular function as an index for cardiovascular disease. This protocol directly measures the stress and strain on an arterial vessel as an index of vascular function.</div><div class = "text-block"><span style = "font-weight:bold;">Modified from: </span><span>Symons et. al. Arterioscler Thromb Vasc Biol. 2002 May 1;22(5):772-80.</span></div></div>

[STEPS]
?. Mice are anesthetized with an intraperitoneal injection with 50 mg pentobarbital/kg weight.
?. Either common carotid arteries or aorta are dissected as described in Macrovascular Permeability and Lipoprotein Flux protocol. Artery explants can be: a. Immediately utilized for analysis of vascular stiffness b. Stored in Krebs-Henseleit at 4°C overnight.c. Snap-frozen in liquid nitrogen and stored at -80°C, samples will be thawed overnight in Krebs-Henseleit at 4°C prior to analysis.
?. Two stainless steel rods (200-µm outside diameter) qre inserted through the lumen of a 1-mm section of carotid artery in a parallel manner while the vessel is immersed in Krebs-Henseleit buffer. One rod is attached to a motorized controller, while the other to a force transducer. (As the motorized controller pulled the rods apart, the vessel tension is recorded.)
?. Arterial segments are preconditioned three times at [almost equal to] 10% of their maximal load.
?. Stress (vessel tension development divided by vessel area, N/mm²) versus strain (vessel circumference) curves is generated so that maximum strain (ie, strain at vessel breakage), maximal stress (vessel tension at maximum strain), and failure energy (area under the stress-strain curve) can be calculated. a. MPS is defined as the maximal slope of the maximal slope of the load vs. strain relationship.
?. Experiments are performed on three 1-mm segments of each artery, and the results are averaged.
TITLE: Agarose gel electrophoresis
AUTHORS: Yuichiroh  Ikagawa

[DESCRIPTION]
Electrophoresis is the most basic technique for identifying DNA amplified by PCR or restriction enzyme treatment. Agarose gels are used to separate the DNA by molecular weight. Here we describe the technique using a 1.5 % agarose gel.

[STEPS]
1. Weigh out 0.75 g of agarose powder and add it to the triangular flask.
2. Add 49 ml of sterile water to the first flask and shake gently.
3. Add 1 ml x50 TAE buffer to the flask and heat in the microwave.
4. Allow the melted agarose to cool for 5 minutes before pouring it into a template set with a comb.
5. Cover with plastic wrap and leave for 15 minutes until the gel has solidified.
6. Mix 1 ul of loading dye with 5 ul of sample DNA.
If necessary, the sample DNA solution can be diluted and added.
7. Carefully apply 6 ul of the DNA-dye mixture to the wells.
8. Run the electrophoresis 100 volts for 30 minutes.
9. Remove the gel from the electrophoresis bath and stain with ethidium bromide (EtBr) for 20 minutes.
10. Check the stained DNA with a transilluminator.
TITLE: Hydrochloric Acid-Potassium Chloride Buffer (HCl-KCl)
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.</div></div>

[STEPS]
?. [Hydrochloric Acid-Potassium Chloride Buffer (HCl-KCl)]
Mix  and indicted volume of hydrochloric acid.
pH Range:   to  (a) 0.1 M Potassium chloride: 7.45 g L-1 (M.W.: 74.5 g mol-1)(b) 0.1 M Hydrochloric acid
[potassium chloride]
?. [Hydrochloric Acid-Potassium Chloride Buffer (HCl-KCl)]
Mix and adjust the final volume to  with deionized water.
100 ml
?. [Hydrochloric Acid-Potassium Chloride Buffer (HCl-KCl)]
Adjust the final pH using a sensitive pH meter.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is to further process the sequences generated from ICE and Quiver.</p>

[BEFORE_START]
<p>Raw data from Pacbio Iso-Seq needs to be processed with RS IsoSeq (version 2.3) pipeline.</p>

[GUIDELINES]
<p><strong>Remove of the detected contaminant sequences</strong></p>
<p>If X &gt; Y, then the sequences were removed. This is done subsequently after each detecting steps, therefore, the dataset number is decreased and its name has changed from step 4 onwards.</p>

[STEPS]
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TITLE: Transformation using Heat-Shock
AUTHORS: Alba Balletbó

[STEPS]
?. Add plasmid DNA (1 µL of miniprep/2 µL of ligation mixture/...µL of cesium-purified DNA).
?. Incubate on ice for 30 minutes.
?. Heat shock at exactly 42ºC for exactly 1:30 minutes, then chill on ice again for 5 minutes.
?. Culture in 1 mL SOC for 1 hour at 37ºC and pre-warm the agar plates in the meantime.
?. Spin the culture down at 3000rpm for 3 minutes, discard 900 µL and resuspend the pellet in the remaining 100 µL.
?. Plate accordingly on agar plates with the appropriate antibiotic, if applicable.
?. Repeat Step 3, once.
TITLE: Capture of Philaenus spumarius and Cicadella viridis
AUTHORS: Niels Appelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to capture spittlebugs (Philaenus spumarius) and Green Leafhoppers (Cicadella viridis)</div></div>

[STEPS]
?. It is recommended to familiarize yourself with the general characteristics of Spittlebugs and Green leafhoppers prior to attempting to capture the insects.
?. Find a terrain in your vicinity with short (under 30 cm) herblike plants. No one source can be named, as both species are extremely polyphagous.
?. Swipe an insect net through vegetation in one continuous motion. It is recommended to take large steps while sweeping, as insects flee areas where swiping occurs.
?. Tap the net at insects that are suspect of being the target insects. Insects that respond by flying are very unlikely to be spittlebugs or leafhoppers. Note that both target insects have very short antenna.
?. Target insects are captured with a suction exhauster (e.g. https://www.vermandel.com/product/entomologie-producten/vangen/insectenvallen/verzamelzuigexhauster/ )
 Note: Vermandel recommends the use of white capped 20 mL tubes as temporary storage tube, but 50mL conical centrifuge tubes are preferable because they are deeper, and therefore their usage prevents insects from crashing into the bottom of the tube and being damaged.Note: prevent the inside of the storage container from getting wet, especially in case of capture during rain, as insects can easily get caught in droplets due to surface tension and drown or be damaged.
?. The temporary storage tubes are inverted, and insects are allowed to climb upwards.
?. The temporary storage tube is held sidewards while the suction exhauster is removed and the cap is screwed onto the tube. Holding tubes sideward significantly lowers the chance of insects escaping.
?. Within 2 hours, move the insects from the temporary storage tubes into containers suitable for long term storage or directly into an experimental setup. This is important because these insects are ill-equipped to survive in conditions of low humidity and/or prolonged starvation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<strong>Viral Bioinformatic Resource Centre</strong>
<ul>
<li><strong>Provide databases of viral genomic information.</strong>
<ul>
<li>Please check the <strong><em>Organisms</em></strong> menu to see which viruses we support: we’re now focusing on large DNA viruses</li>
<li>The VOCs (Virus Orthologous Clusters) database is at the heart of our system.</li>
<li>The database links directly to integrated tools for comparative analyses.</li>
<li>VOCs sorts genes into ortholog clusters (e.g. RNA polymerase) to simplify data retrieval.</li>
</ul>
</li>
<li><strong>Provide easy access to the genes, gene families, and genomes of the different virus families.</strong>
<ul>
<li>via a unique series of powerful Java tools that support multiple computer platforms (see <strong><em>VBRC Tools</em> </strong>menu).</li>
<li>design and build software to tackle specific bioinformatics/virology problems, often in collaboration with virologists.</li>
</ul>
</li>
<li><strong>Rally the research community to provide expert curation of these viral genomes by:</strong>
<ul>
<li>Adding value to GenBank sequences through enhancing and updating genome annotations</li>
<li>Linking to research reviews/papers for the research community.</li>
</ul>
</li>
<li><strong>Collaborate with researchers to help on specific bioinformatics problems, e.g.</strong>
<ul>
<li>Custom searches of the databases</li>
<li>Building new features into our tools</li>
<li>Help with genome annotation</li>
</ul>
</li>
</ul>

[STEPS]
?. 
?.
TITLE: Cell Harvesting Protocol
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A general procedure for harvesting bacterial cells for flow cytometry or fluorescence microscopy</div><div class = "text-block">Write up by S. Shore 11/17/2020 based on B. Bogati communications, proofread by Bikash Bogati</div></div>

[STEPS]
?. [Procedure]
Dilute the overnight culture to OD600 0.01 and grow cells in an appropriate medium to the desired time point/OD600 in at least 10mL of liquid culture.
?. [Procedure]
Harvest cells directly into pre-labeled falcon tubes (if running flow same-day) or Eppendorf tubes. Once cells have been harvested, minimize exposure to direct light if looking for fluorescence. For E. coli from overnight LB, at least 25uLFor E. coli from exponential phase (OD~0.3) LB, at least 50uLFor E. coli from the overnight or exponential phase in minimal media, at least 500uL
?. [Procedure]
For membrane depolarization assay:Wash the cells twice with 1x PBS and resuspend in 1 ml PBSAdd DiBAC4-3 reagent- final concentration 10mg/mL (4ml of 2.5 mg/mL working stock in 1 mL)Mix and incubate in dark for 20 minutes. Wash twice in PBS.Fix using paraformaldehyde and glutaraldehyde (see below)
?. [Procedure]
Optional: Fix with desired fixation method in the chemical fume hoodRequired if using the LSR-II in Mossman. Special permission needed to run live-cells through a flow cytometer. Potential fixation methods:0.5% paraformaldehyde for 10 min in a cold room in dark (T. Hancock)3% glutaraldehyde for 1hr at room temp in dark (R. Johnston) paraformaldehyde to final concentration 2.8% (vol/vol) and glutaraldehyde to 0.04% (vol/vol) for 15 min at room temp in dark (Dr. J.Mannik)
?. [Procedure]
Wash cells 3 timesCentrifuge at 4 degrees until pellets form (tabletop for 3 min, if in falcon tubes, 10 min in a large centrifuge)Decant supernatant in an appropriate chemical waste container in the hoodFlood sample with PBS and vortexRepeat steps 1-3 2 additional times
?. [Procedure]
Decant liquid and add PBS to the desired volume.For flow and E. coli, 1 mL for minimal media samples, 0.5 mL for LB samples if the followed recommendation in step 2.If mounting cells on slides for microscopy, do not add PBS during the last round. Aspirate remaining liquid out instead.
?. [Procedure]
If applicable, store cells in the fridge (4 degrees).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em>Listeria monocytogenes</em> is a bacterial pathogen that enters and proliferates in the cytosol of mammalian cells. Following entry, bacteria disrupt the invasion vacuole and reach the cytoplasm, where they replicate and use the actin cytoskeleton to propel themselves from cell to cell [1, 2]. To achieve this cytosolic lifestyle, <em>Listeria </em>deploys virulence effectors that hijack diverse cellular processes [3, 4]. Human epithelial cells grown <em>in vitro</em> can be used to study the <em>L. monocyt</em>ogenes infectious process. However, rapid multiplication and dissemination of bacteria may induce cell death and detachment, thereby forming lytic plaques. Thus, <em>in vitro</em> infections with <em>L. monocytogenes</em> have been restricted to short time courses (usually from a few minutes to one day). In order to study <em>L. monocytogenes</em> long-term infections, we have set up a protocol, with several modifications to the gentamicin protection assay previously used for short-term infections with this pathogen [5]. In a subset of human cells, such as hepatocytes or trophoblast cells, this protocol enables to observe and study the entrapment of <em>L. monocytogenes</em> within vacuoles, termed “Listeria-Containing Vacuoles” (LisCVs), after the intercellular dissemination phase of <em>Listeria</em>.</p>
<p> </p>
<p>Here, this protocol describes the assays used to study LisCVs in a JEG3 trophoblast cell monolayer.</p>

[STEPS]
TITLE: Capture RBP -RNA Complexes on Beads
AUTHORS: Eric L. Van Nostrand, Thai B. Nguyen, Chelsea Gelboin-Burkhart, Ruth Wang, Steven M. Blue, Gabriel A. Pratt, Ashley L. Louie, Gene W. Yeo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Profiling of RNA binding protein targets in vivo provides critical insights into the mechanistic roles they play in regulating RNA processing. The enhanced crosslinking and immunoprecipitation (eCLIP) methodology provides a framework for robust, reproducible identification of transcriptome-wide protein-RNA interactions, with dramatically improved efficiency over previous methods. Here we provide a step-by-step description of the eCLIP method, along with insights into optimal performance of critical steps in the protocol. In particular, we describe improvements to the adaptor strategy that enables single-end enhanced CLIP (seCLIP), which removes the requirement for paired-end sequencing of eCLIP libraries. Further, we describe the observation of contaminating RNA present in standard nitrocellulose membrane suppliers, and present options with significantly reduced contamination for sensitive applications. These notes further refine the eCLIP methodology, simplifying robust RNA binding protein studies for all users.</div></div>

[STEPS]
?. [Couple Antibody to Magnetic Beads (Start During Cell Lysis)]
Use  +  per sample.
[beads]
[antibody]
?. [Prewash Beads]
Magnetically separate beads, remove the supernatant.
?. [Prewash Beads]
Wash beads 2× in .
[cold lysis buffer]
?. [Prewash Beads]
Resuspend beads in .
[cold lysis buffer]
?. [Bind Antibody]
Add  to  washed beads.
[antibody]
100 µl
?. [Bind Antibody]
Rotate at  for .
0 Room temperature
?. [Capture RBP -RNA Complexes on Beads]
Wash antibody beads 2× in .
[cold lysis buffer]
?. [Remove Input Samples]
To a new tube, take  for Preparative gel, store at .
[(2%) of total beads + lysate sample]
4 °C
?. [Remove Input Samples]
To a new tube, take  for Imaging gel, store at .
[(2%) of total beads + lysate sample]
4 °C
?. [Capture RBP -RNA Complexes on Beads]
Add the supernatant from Step 9 of Cell Lysis and Fragmentation to washed antibody beads.
?. [Capture RBP -RNA Complexes on Beads]
Rotate at .
4 °C
TITLE: Histological Evaluation of Renal Fibrosis in Mice
AUTHORS: Daisuke Katagiri, Takamune Takahashi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary </span></div><div class = "text-block">This protocol describes a protocol to evaluate histological fibrosis in mouse kidney. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/biem4kcn.jpg" /></div><div class = "text-block"><span style = "font-weight:bold;">References </span></div><div class = "text-block"><span>1. Katagiri D, Hamasaki Y, Doi K, et al. Interstitial renal fibrosis due to multiple cisplatin treatments is ameliorated by semicarbazide-sensitive amine oxidase inhibition. </span><span style = "font-style:italic;">Kidney Int 2015; 89: 374 - 385. </span></div><div class = "text-block"><span>2. Hara S, Umeyama K, Yokoo T, et al. Diffuse glomerular nodular lesions in diabetic pigs carrying a dominant-negative mutant hepatocyte nuclear factor 1-alpha, an inheritant diabetic gene in humans.</span><span style = "font-style:italic;"> PLoS One 2014; 9: e92219. </span></div></div>

[STEPS]
?. Tissue stain a. picrosiriusred stain  Collagen including collagenand III were stained with the Picrosirius Red Stain kit (Polysciences Inc., PA, US) according to the manufacturers’ protocol.11. Dip in Xylene 5 min x 2 times 2. Dip in 100 % EtOH 5 min x 2 times 3. Dip in 90 % EtOH 5 min 4. Dip in 80 % EtOH 5 min 5. Dip in 70 % EtOH 5 min 6. Dip in 50 % EtOH 5 min 7. PBS wash 5 min x 3 times 8. Solution A 2 min 9. PBS wash 5 min x 3 times 10. Solution B 60 min 11. Solution C 2 min 12. 70 % EtOH 45 sec 13. Dehydration, Cleaning, Mount b. Collagen IV immunostain For Collagen IV staining, slides were placed on the Bond Max immunohistochemistry (IHC) stainer (Leica Biosystems., IL, US). Antibodies against primary collagen IV (Abcam plc., Cambridge, UK) 2  diluted 1:600 were applied to sections and incubated for one hour. All procedure was carried out at Translational Pathology Shared Resource at Vanderbilt University Medical Center.
?. Computational QuantificationThe Bond Refine Polymer detection system (Leica Biosystems.) was used for visualization. Region of interest (ROI) was manually defined as cortical region, which is combined cortex and outer stripe of outer medulla (OSOM). The Picrosirius red- (red) and collagen IV-positive areas (dark) in cortex were calculated using image analysis software (Digital Image Hub; Leica Biosystems.) with unified threshold respectively. The regional fibrosis index was estimated by the area percentage of the positive pixels. Analyses were performed for 6 - 7 sections of each kidney (12 – 14 sections per mouse).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
More than 40 years ago, Safferman and Morris (1963) used the plaque assay method to isolate the first cyanophage that infects a freshwater filamentous cyanobacterium, <em>Plectonema boryanum</em>. Since then, this approach has been used successfully to detect and isolate a number of different phages infecting marine<em> Synechoccocus</em> and <em>Prochlorococcus</em> (e.g., Suttle and Chan 1993; Waterbury and Valois 1993; Wilson et al. 1993; Sullivan et al. 2003).<br /><br />The advantages of this method are that results are easily interpreted as plaques formed on pigmented lawns can be easily identified. Since a plaque is the result of a single infection event, the virus can be easily purified and cloned. The first part of the procedures described as follows: (1) preparation of base plates and (2) preparation of top agar/agarose are applicable also for bacteriophage plaque assays.

[GUIDELINES]
For example, purified agar or agarose (1% w/v) is added to your media of choice and autoclaved. This will provide a support base for the top agar/agarose overlay as well as nutrients for the host cells. For best results, use plates within 1 week of pouring.<br /><br />Tip: plates can be fast-tracked: dry plates at 37°C; leave lids slightly ajar; monitor closely to prevent over drying.<br /><br />Considerations: Depending on the composition of the media used, the addition of solidification agents (in particular the combination of high salinity seawater-based media and common agar such as Bacto Agar) can often result in the formation of precipitates when autoclaved together. These flocks can sometimes interfere with interpretation of the plaque assay. Moreover, impurities in common agar can negatively affect the growth of the host cells. Here are some suggestions on how to reduce the formation of these precipitates. Some testing may be required to determine the best combination to use for your particular situation. <br /><br />a. Do not use common agar; rule of thumbthe whiter the agar, the cleaner it is. <br />b. Use commercially available purified agar or agarose; or clean common agar using a washing procedure such as the one outlined in Waterbury and Willey (1989). <br />c. Reduce the salinity of seawater media with purified water; e.g., to 20-25 psu. <br />d. Add purified agar or agarose to autoclaved media aseptically and then melt the agar/agarose in the microwave (bring to a short boil 2-3 times to completely dissolve the agar/agarose). <br />e. For cells that will grow in artificial media, prepare media and gelling agent at 2x concentration and autoclave separately. When cooled to ca. 60°C, gently mix the gelling agent into the media and dispense immediately. <br />f. In the case of artificial media, add agar/agarose to filter-sterilized media and melt the gelling agent in the microwave

[STEPS]
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