eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Version 3<br /><br />This protocol describes the use of Cesium Chloride to purify viruses with an optional dialysis step to remove the CsCl. DNase I treatment, CsCl purification, and sucrose purification methods were compared using replicated viral metagenomics in Hurwitz et al. 2012.<br /><br /><span style="text-decoration: underline;"><strong>References:<br /></strong></span>Hurwitz, B.L., Deng, L., Poulos, B.T., &amp; Sullivan, M.B. (2012). Evaluation of methods to concentrate and purify ocean virus communities through comparative, replicated metagenomics. Environ Microbiol. 15(5), 1428&ndash;1440. doi:10.1111/j.1462-2920.2012.02836.x.

[GUIDELINES]
This protocol is part of a larger collection of Cesium-Chloride related protocols. This is number (4) of (4):<br />1. <a href="https://www.protocols.io/view/Cesium-Chloride-gradients-and-DNA-Extraction-Virus-c7dzi5" target="_blank">Cesium Chloride Gradients</a><br />2. <a href="https://www.protocols.io/view/CsCl-Step-Gradient-to-Purify-Phage-c4zyx5" target="_blank">CsCl Step Gradient to Purify Phage</a><br />3. <a href="https://www.protocols.io/view/DNA-Extraction-of-Viruses-using-Wizard-Prep-Column-c26yhd" target="_blank">Cesium Chloride and DNA Extraction of Viruses using Wizard Prep Columns</a><br />4. <a href="https://www.protocols.io/view/Cesium-Chloride-Dialysis-for-Viruses-c7jzkm?guidelines" target="_blank">Cesium Chloride Dialysis for Viruses<br /><br /></a>To prepare a CsCl solution of a particular density, the percent by weight of CsCl can be calculated by the formula:<br />% wt/wt = 137.48 - 138.11/p<br />where 'p' is the desired density. For example, for p = 1.7 g/mL, use 56.24g CsCl and 43.76 mL H2O.<br /><br />Needed:<br />
<ul>
<li>Milli-Q Water</li>
<li>MTN 100 (NaCl, Tris-Cl (pH 7.5), MgCl2)</li>
<li>Slide-A-Lyzer dialysis casettes</li>
<li>Tris Base</li>
</ul>
<br />If it is necessary to remove the CsCl, the virus preparation should be dialyzed against a buffer containing sodium chloride and magnesium chloride. The virus should be dialyzed against 4 changes of buffer with the first at a high salt concentration, then at an intermediate salt concentration and finally twice using the final salt concentration. Dialysis should be at 300x the sample volume for 30 min at room temperature. The dialysis membrane should have a 10,000 molecular weight cut-off (10K MWCO).

[STEPS]
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TITLE: Counting worms / C. elegans / nematodes
AUTHORS: Cristian Riccio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Count worms, e.g. arrested L1s after an overnight starvation in M9 on a rotating wheel.</div></div>

[STEPS]
?. Shake your wormy solution to make the concentration of worms in it more homogeneous.
?. Pipet 10 microlitres of wormy solution in a streak onto a glass slide. Don't pipet the 10 ul in a blob. That would make it harder to count.
?. From left to right, or right to left, follow the streak under the microscope and count the worms. For this, you can use a manual click counter to make it easier. You should aim to count between 50 and 150 worms. If the number of worms in 10 ul is higher than this, make a dilution of the wormy solution to achieve between 50 and 150 worms in 10 ul. If the number of worms is less than 50 per 10 ul, consider increasing the volume counted.
?. Count another two slides and take the arithmetic mean. You can then infer the concentration of worms for any volume. The inferred concentrations from the three replicates give you an idea about the range within which the true concentration of worms lies.
TITLE: MicroCT protocols for scanning and 3D analysis of adult Hexaplex trunculus
AUTHORS: Eva Chatzinikolaou, Kleoniki Keklikoglou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Micro-computed tomography (micro-CT) is a high-resolution 3D-imaging  technique which is now increasingly applied in biological studies  focusing on taxonomy and functional morphology. The creation of virtual  representations of specimens can increase availability of otherwise  underexploited and inaccessible samples. This protocol aims to standardise micro-CT scanning and 3D analysis for the marine gastropod species </span><span style = "font-style:italic;">Hexaplex trunculus. </span></div></div>

[STEPS]
?. [Sample preparation]
Anesthetization of gastropods with 7% MgCl2
?. [Sample preparation]
Storage in -20°C
?. [Sample preparation]
Sample placement inside a custom-made felisol holder without any scanning medium (in the air)
?. [microCT scanning]
?. [Images reconstruction]
Projection images were reconstructed into cross sections using the SkyScan’s NRecon software (Bruker, Kontich, Belgium)
?. [Images reconstruction]
Upload projection images
?. [Images reconstruction]
Perform X-Y alignment
?. [Images reconstruction]
Reconstruction parametersSmoothing: 2Ring artifact correction: 20Beam hardening correction: 59%Attenuation coefficients: 0 - 0.13Save as: 16-bit TIFF images
?. [Image analysis]
3D Analysis using the software CT Analyser v.1.18.4.0 (CTAn, Bruker, Kontich, Belgium)
?. [Image analysis]
Upload reconstructed images
?. [Image analysis]
Creation of a Region of Interest (ROI)
?. [Image analysis]
Binary thresholding with a grayscale histogram range 30 - 255 for measuring relative shell density
?. [Image analysis]
Custom processing plugina) Thresholding: 30-255b) Despeckle: remove black speckles c) Despeckle: sweep all except the largest objects
?. [Image analysis]
3D Analysisa) Structure thickness: average value of the diameters of the largest spheres which can be fitted in each point of the shell structure (“sphere-fitting” method)b) Closed shell porosity: total number of closed pores and % of the closed porosity of the shell (i.e. total volume of enclosed pores of each specimen as a % percentage of the total shell volume)
?. [Image analysis]
Save colour coded images for visualisation of the shell structure thichness using the CTVox software (Bruker, Kontich, Belgium)
?. [microCT scanning]
Scanning parameters for adult Hexaplex trunculusVoltage: 100 kVCurrent :100 μAFilter: aluminium and copper Pixel size: 13.79 μmCamera binning: 2 × 2Exposure time: 2480 msRotation: 180°Rotation step: 0.60ºFrame averaging: no
TITLE: Early versus delayed feeding after endoscopic submucosal dissection: a systematic review and meta-analysis (protocol)
AUTHORS: Jun Watanabe, Joji Watanabe, Kazuhiko Kotani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Background</span><span>: Endoscopic submucosal dissection (ESD) is increasingly performed worldwide due to its safety and effectiveness. This study will aim to assess the evidence of early versus delayed feeding after ESD on quality of care, which remains to be fully determined.</span></div><div class = "text-block"><span style = "font-weight:bold;">Methods</span><span>: Electronic database (PubMed, the Cochrane Central Register of Controlled Trials, EMBASE) and the trial registry (the World Health Organization International Clinical Trials Platform Search Portal and ClinicalTrials.gov) wii be searched. Study selection, data abstraction and quality assessment will be independently performed using Grading of Recommendations Assessment, Development and Evaluation approach. Self-rated satisfaction and hospital stay will be chiefly analyzed.</span></div></div>

[STEPS]
?.
TITLE: Cerulenin experiment
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Rescue from Cerulenin Inhibition</div><div class = "text-block">Based on Saito, et al., 2018</div></div>

[STEPS]
?. [Steps]
Grow strain of interest overnight in 5 ml BHI
?. [Steps]
The next day, set up the following tubes:Tube with no ceruleninTube with cerulenin + solvent control (for fatty acid)Tube with cerulenin + fatty acid of interest
For current studies, use 5mg/ml of cerulenin (note, can dissolve stock in 10 mg/ml ethanol) and 5ug/ml of fatty acid interested in testing.Perform in glass tubes with either 5 or 10 mL total volume
?. [Steps]
In am, dilute the strain to be an OD600 nm of 0.01 into each of the above tubes.  This is time “0”
?. [Steps]
Record the OD600 nm at time 2, 4, 6, and 24 hours
?. [Steps]
Graph the results, making sure to add temporal separation in the graphs for the 24 hour time point.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em>The MELD Project is an international collaboration aiming to create open-access, robust and generalisable tools for FCD detection. To this end, we will train a neural network classifier on MRI features from FCD patients from multiple centres worldwide.</em></p>
<p><strong> </strong></p>
<p><strong>Protocol 2 provides instructions on how to create FreeSurfer cortical segmentations.</strong></p>
<p> </p>
<p>These instructions are based on the freely available protocols on the ENIGMA-epilepsy website http://enigma.ini.usc.edu</p>
<p> </p>
<p>We are very grateful to Derrek Hibar, Neda Jahanshad, Roberto Toro, Jerod Rasmussen, Theo van Erp who wrote the orginal ENIGMA protocols and offered them with an unlimited license without warranty! </p>
<p> </p>
<p>The main changes are the paths, which are now directed to the meld folder.</p>
<p> </p>
<p> </p>

[BEFORE_START]
<p>Ensure that you have "cloned" the MELD file structure from github. (https://github.com/MELDProject/meld)</p>
<p> </p>
<p>To do this, in a terminal window cd into the location you wish to store the data.</p>
<p>`cd &lt;path&gt;`</p>
<p>Then "clone" the repository using the following command:</p>
<p> </p>
<p>git clone https://github.com/MELDProject/meld</p>
<p> </p>
<p>This will download all of the scripts, template folder structure and template control data necessary for the MELD preprocessing.</p>
<p> </p>
<p>This contains the MELD_site_code_participants.csv file that is required for this protocol.</p>
<p> </p>
<p><strong>If you have any questions or run into problems, please feel free to contact the MELD project: (meld.study@gmail.com)</strong></p>

[GUIDELINES]
<p>These instructions are based on the freely available protocols on the ENIGMA-epilepsy website http://enigma.ini.usc.edu/ongoing/enigma-epilepsy/enigma-epilepsy-protocols/</p>
<p> </p>
<p>We are very grateful to Derrek Hibar, Neda Jahanshad, Roberto Toro, Jerod Rasmussen, Theo van Erp who wrote the orginal ENIGMA protocol. The main changes are the paths, which are now directed to the meld folder.</p>

[STEPS]
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TITLE: SPARC Cat - Sham Control Chronic Cat 2, Day 14
AUTHORS: Brett     Hanzlicek, Anna Rietsch, Margot Damaser

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a procedure for a sham control chronic cat experiment (Day 14) for cystotomy (bladder surgery).  The cystotomy is performed without UroMOCA implantation.  The cat is observed daily and imaged on day 0, 14 and 30 to track changes to the bladder and overall cat health in response to the cystotomy.  This protocol includes urodynamics and imaging for Day 14 in the chronic experiments.</div></div>

[STEPS]
?. [Transport Cat]
Transport cat from housing site to surgery site.
?. [Animal Prep and catheter placement]
Animal is anesthetized and abdomen is shaved by the vet team.  The cat is then moved into the surgery room and attached to monitors by the vet team.
?. [Animal Prep and catheter placement]
Drape animal and perform betadine scrub on abdomen and genitals.
?. [Animal Prep and catheter placement]
Put surgilube on 3.5Fr catheter and insert into bladder through the urethra.  Advance the catheter until resistance is met, then pull back 2-3cm.
?. [Animal Prep and catheter placement]
Use gentle suction with syringe to withdraw urine from bladder through the catheter.  Measure the volume and save the urine for urinalysis.  urine removed
6 mL
?. [Cystogram - DYNA CT]
Use 1:5 dilution of contrast to saline to visualize bladder.Fill the bladder with   contrast and take a single CT image.Add   more to bladder (total of 15ml) and take single CT imageTake a 3D CT image with 15ml contrast:saline in the bladder
10 mL
5 mL
?. [Cystogram - DYNA CT]
Empty Bladder
?. [Urodynamics/Cystometry 1]
Performed after cat is transferred from Isoflurane to propofol anesthetic.
Fill rate is 2ml saline/minute using syringe pump.
Data is recorded using Labview software.
Pressure is recorded using an external pressure transducer connected to syringe pump on one end and tubing that leads to the catheter on the other end.
?. [Urodynamics/Cystometry 1]
Fill bladder with saline and record pressure using pressure transducer connected to  LabViewFill to  ;End round 1 urodynamicsTug on catheter after filling to visualize contraction in Labview software
15 mL
?. [Urodynamics/Cystometry 1]
Empty Bladder
?. [Urodynamics/cystometry 2]
Fill bladder with saline at 2ml/min
?. [Urodynamics/cystometry 2]
Fill to  ; End round 2 urodynamics
15 mL
?. [Wake and return cat]
Wait for cat to wake up and return to housing site.
TITLE: Hydrophobicity Protocol
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">P-Xylene Hydrophobicity Assay</div><div class = "text-block">Note that I have written this before doing it. Changes most likely will be made.</div></div>

[STEPS]
?. [Steps]
Grow 10mls OG1RF in BHI using short-term or long-term supplementation
?. [Steps]
At 0.3 OD for long-term or 30 minutes after supplementation for short-term, spin cells and wash twice using PUM buffer
?. [Steps]
Aliquot 1 ml 6 times into glass tubes and mix with a range of P-xylene volumes
?. [Steps]
Vortex vigorously for 30 seconds or place in a shaker for 2 minutes (for many samples)
?. [Steps]
Incubate static at 37oC for 15 minutes
?. [Steps]
Carefully extract 200µl of the aqueous phase and add to 800µl PUM buffer in a cuvette
?. [Steps]
Record OD600 for each P-xylene xoncentration
?. [Steps]
Present each sample s a percentage of cells in the aqueous phase relative to PUM buffer with no p-xylene
?. [Steps]
OD600nm Xµl p-xylene/ OD600nm PUM only = % cells in aqueous phase
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol gives an introduction to running VirSorter in the iPlant Discovery Environment.

[BEFORE_START]
To access VirSorter on iPlant, you will first need to create a (free) account. This can be done at this address: <a href="https://user.iplantcollaborative.org/register/" target="_blank">https://user.iplantcollaborative.org/register/</a> <br />Help can found at: <a href="https://pods.iplantcollaborative.org/wiki/display/start/Registering+for+and+Managing+your+iPlant+Account" target="_blank"> https://pods.iplantcollaborative.org/wiki/display/start/Registering+for+and+Managing+your+iPlant+Account</a>

[GUIDELINES]
See the wiki page of VirSorter for a more complete descripton of the result files:<br /><br /><a href="https://pods.iplantcollaborative.org/wiki/display/DEapps/VIRSorter+1.0.2" target="_blank">https://pods.iplantcollaborative.org/wiki/display/DEapps/VIRSorter+1.0.2</a><br /><br />

[STEPS]
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TITLE: Plaque PCR
AUTHORS: Jiaxin Li

[STEPS]
?. Mark independent plaques on the back of the Petri dish with a black pen. Take 6 points in a plate and try to take points randomly and evenly.
?. Take 24 1.5 ml EP tubes.A) Add 200 μl SM Buffer per tubeB) Add 2 μl chloroform to each tube
?. [Sampling]
Suck out the plaque just spotted with the pipette
?. [Sampling]
Put into the prepared EP tube.
?. [Sampling]
Open the metal bath to 95 degrees for lysis of the phage
?. [Sampling]
Place the EP tube at room temperature for one hour to dissolve the sample sufficiently and mix several times during the period.
?. [PCR system]
KOD FX 20ul system
?. [PCR system]
Add 18.4 μl to each tube (1.6 μl template is needed)
?. After an hour, put the EP tube in a metal bath and heat it for 10 minutes.
?. Centrifugation for 5 min, 12,000 rpm
?. Take 1.6ul supernatant from EP tube and add it to new EP tube.
?. Centrifuge the EP tube briefly with a centrifuge.
?. Place in PCR instrument.
TITLE: Membrane and cytosol fractionation
AUTHORS: wusj, schekman

[DESCRIPTION]
This protocol describes membrane and cytosol fractionation of cells expressing different DNAJC5 isoforms

[STEPS]
SECTION: Cytosol fractionation
1. Cells (one 10 cm dish) were cultured to 70% confluence and transfected with different constructs
of DNAJC5
SECTION: Cytosol fractionation
2. One day after transfection, we harvested the transfected cells by scraping in 1 ml B88 (20 mM HEPES-KOH, pH 7.2, 250 mM sorbitol, 150 mM potassium acetate, and 5 mM magnesium acetate) plus a cocktail of protease inhibitors
SECTION: Cytosol fractionation
3. Cells were homogenized by 10 passages through a 22G needle
SECTION: Cytosol fractionation
4. Homogenates were centrifuged at 500×g for 10 min and the resulting post-nuclear supernatant (PNS) fractions were centrifuged at 100,000×g for 90 min

SECTION: Cytosol fractionation
5. High-speed supernatant fractions were then subjected to a repeat centrifugation to achieve a clarified cytosol fraction
SECTION: Cytosol fractionation
6. The pellet fraction was washed and resuspended in the same volume of B88
SECTION: Cytosol fractionation
7. Resuspended material was also centrifuged again to collect a washed membrane fraction
SECTION: Membrane fractionation
9. The PNS was subjected to differential centrifugation at 3000×g for 10 min 

SECTION: Cytosol fractionation
8. Membranes were lysed in lysis buffer
SECTION: Membrane fractionation
10. The supernatant was centrifuged at 25,000×g for 20 min

SECTION: Membrane fractionation
11. The supernatant was centrifuged at 100,000×g for 30 min

SECTION: Membrane fractionation
12. Membrane fractions were normalized to phosphatidylcholine content and analyzed by immunoblot
SECTION: Proteinase K protection assays
13. The 25,000×g membrane fraction was aliquoted into three tubes: one without proteinase K, one with proteinase K (10 μg/ml), and one with proteinase K plus TritonX-100 (0.5%)
SECTION: Proteinase K protection assays
14. The incubation was conducted on ice for 20 min

SECTION: Proteinase K protection assays
15. The reaction was stopped by sequential addition of PMSF (1 mM)
SECTION: Proteinase K protection assays
16. Add sample buffer. Then, samples were then heated on metal block at 95 °C for 5 minand analyzed by SDS-PAGE and immunoblot.
TITLE: Yale - Total Cholesterol
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to determine the total concentration of cholesterol present in blood, serum, and plasma. Cholesterol esters are hydrolysed by cholesterol esterase. Cholesterol is then oxidized by cholesterol oxidase with formation of hydrogen peroxide. Peroxidase uses the hydrogen peroxide, phenol, and 4-aminotipyrine to form a quinoneimine dye which is measured at 500 nm.</div></div>

[STEPS]
?. Calibrate Cobas for Total Cholesterol analysis by running a multi analyte standard and two control serum.
?. Sample handling as performed by Cobas Mira Plus.     a) Pipette 3 µL of sample into a cuvette slot.    b) Add 275 µL of Cholesterol Rapid Liquid Reagent.    c) Mixture is incubated at 37ºC for 10 minutes.    d) Absorbance is measured at 500nm.
TITLE: Forebrain neural progenitor cells (fbNPCs) differentiation from hiPSCs (dual SMAD inhibition)
AUTHORS: anita.adami

[DESCRIPTION]
This protocol described how to differentiate hiPSCs to forebrain neural progenitor cells using dual SMAD inhibition

[STEPS]
SECTION: hiPSCs dissociation
1. Human induced pluripotent stem cells (hiPSCs) are rinsed once with DPBS (GIBCO) and dissociated when 70-90% confluent with 0.5 mM EDTA (75 ml/cm2; GIBCO) at 37 °C for 7 min 

SECTION: hiPSCs dissociation
2. Dissociated cells are then carefully washed away from the wells and collected in wash medium (9.5 mL DMEM/F-12 (31330-038; GIBCO) and 0.5 mL knockout serum replacement (GIBCO)).

SECTION: hiPSCs dissociation
3. The cells are then centrifuged at 400 x g for 5 minand resuspended in iPS brew medium (StemMACS iPS-Brew XF and 0.5% penicillin/streptomycin (GIBCO)).

SECTION: hiPSCs differentiation into fbNPCs via dual SMAD inhibition
4. The protocol is based on the one described in Nolbrant et al., 2017 and Grassi et al., 2019.

Day 0 of differentiation. The resuspended cells are then plated on LN111-coated (1.14µg/cm2; Biolamina) coated Nunc multidishes at a density of 10000 cells/cm2 and grown in N2 medium (1:1 DMEM/F-12 (21331020; GIBCO) and Neurobasal (21103049; GIBCO) supplemented with 1% N2 (GIBCO), 2 mM L-glutamine (GIBCO), and 0.2% penicillin/streptomycin). 10 μM of Y27632 (Rock inhibitor; Miltenyi) are also added to the cells when plating. Finally, 10 μM SB431542 (Axon) and 100 ng/ml noggin (Miltenyi) for dual SMAD inhibition are supplemented to the media. 
SECTION: hiPSCs differentiation into fbNPCs via dual SMAD inhibition
5. The media, supplemented with dual SMAD inhibitors, is changed every 2-3 days until day 9 of differentiation. The amount of media is slightly increased at every feeding as the cells' confluency increases. 
SECTION: hiPSCs differentiation into fbNPCs via dual SMAD inhibition
6. Day 9 of differentiation. The cells are fed with N2 media without dual SMAD inhibitors. 
SECTION: hiPSCs differentiation into fbNPCs via dual SMAD inhibition
7. Day 11 of differentiation. The cells are dissociated, centrifuged, and resuspended as described above (hiPSCs dissociation SECTION). They are then replated on LN111-coated Nunc multidishes at a density of 800000 cells/cm2 in B27 medium (Neurobasal supplemented with 1% B27 without vitamin A (GIBCO), 2 mM L-glutamine and 0.2% penicillin/streptomycin Y27632 (10 μM), BDNF (20 ng/ml; R&D), and L-ascorbic acid (0.2 mM; Sigma)). 10 μM of Y27632 are also added. The cells are kept in the same medium until day 14 of differentiation, when they are harvested for downstream analyses (e.g. bulk RNA sequencing).