eamag/protocols-with-steps huggingface dataset:
TITLE: SARS-CoV-2 virus plaque assays [Biosafety Level 3]
AUTHORS: tenOever Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Method to plaque the SARS-CoV-2 virus </div><div class = "text-block">Image attribution:</div><div class = "text-block"><span>Y tambe / CC BY-SA (</span><a href="http://creativecommons.org/licenses/by-sa/3.0/)" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://creativecommons.org/licenses/by-sa/3.0/)</span></a></div><div class = "text-block"><span>Downloaded from: </span><a href="https://commons.wikimedia.org/wiki/File:Plaque_assay_macro.jpg" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://commons.wikimedia.org/wiki/File:Plaque_assay_macro.jpg</span></a></div></div>

[STEPS]
?. [SARS-CoV2 virus plaque assays (Biosafety level 3)]
Seed Vero E6 cells in 6-well plates overnight at  (106 cells/well)
37 °C
?. [SARS-CoV2 virus plaque assays (Biosafety level 3)]
Dilute the SARS-CoV-2 virus logarithmically in infection media
?. [SARS-CoV2 virus plaque assays (Biosafety level 3)]
Replace media with modified DMEM and add diluted virus in each well (200 µl/well)
?. [SARS-CoV2 virus plaque assays (Biosafety level 3)]
Allow virus adsorption for , agitating the plate every .
?. [SARS-CoV2 virus plaque assays (Biosafety level 3)]
Heat the overlay media/oxoid agar mixture to ensure homogenous consistency and add it to each well (2ml/well), incubate at  incubator for .
37 °C
?. [SARS-CoV2 virus plaque assays (Biosafety level 3)]
Fix the plate with 5% formaldehyde (w/ methanol), (1.5 ml/per well)  to ensure virus inactivation and Stain with crystal violet for  to determine viral titers (PFU/ml)
?. [Media Recipes]
Media recipe for modified DMEM: [DMEM with 2% FBS]:   DMEM (w/ L-Glutamate, Sodium pyruvate)  100x P/S 100x Non-Essential Amino Acids (NEAA) Solution 1M HEPES FBS
475 ml
5 ml
5 ml
5 ml
10 ml
?. [Media Recipes]
Media recipe for 2XMEM: Sterile filter with 22 μm filtration unit: 10x MEM 100x P/S 100x L-Glutamine 35%BSA sterile ddH2O 1M HEPES 5% NaHCO3
100 ml
10 ml
10 ml
6 ml
340 ml
10 ml
24 ml
?. [Media Recipes]
Overlay Media (Ensure this is just warm to the touch when added to cells)  PBS 2X MEM Oxoid Agar
1 ml
31.5 ml
17.5 ml
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The protocol was creating adapting components from the manual from Bio-Rad for the analysis software ImageLab. Data analyzed using the protocol here described have appear in the following publications:</p>
<pre>Zeitler AF, Gerrer KH, Haas R, Jiménez-Soto LF. Optimized semi-quantitative blot analysis in infection assays using the Stain-Free technology. J MicrobiolMethods. 2016 Jul;126:38-41. <a href="https://doi.org/10.1016/j.mimet.2016.04.016" target="_blank">doi: 10.1016/j.mimet.2016.04.016</a>. Epub 2016 May 3.PubMed PMID: 27150675.<br />Jiménez-Soto LF, Haas R. The CagA toxin of Helicobacter pylori: abundant production but relatively low amount translocated. Sci Rep. 2016 Mar 17;6:23227. <a href="https://doi.org/10.1038/srep23227" target="_blank">doi: 10.1038/srep23227</a>. PubMed PMID: 26983895; PubMed Central PMCID: PMC4794710.<br />and in the title '<span data-offset-key="2%$Up-0-0">Host cell resistance to CagA translocation is as variable as Helicobacter pylori'. Zeitler et al 2017 accepted in Matters journal.</span></pre>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Described is the procedure used to perform a onestep growth curve for cyanophage BBC1-P1 via plaque assay on <em>Synechococcus</em> sp. BBC1 (Suttle and Chan 1993).

[GUIDELINES]
The procedure is similar to that for bacteriophages with one major difference. Where bacteriophage growth is measurable in the order of minutes, cyanophage growth curves are measured in terms of hours. The burst sizes are similar, being in the tens to hundreds.<br /><br /> As for bacteriophages, infection should be done at a MOI between 0.1 and 0.01. In this instance, the total infective center = the phage input because the proportion of multiplyinfected host cells is small. At higher MOI, the probability of cells infected by more than one virus would increase and the total infective centers (TIC = total number of infected cells + free viruses) become less than the phage input.<br /><br />The dilution factor in going from the adsorption tube to FGT is chosen so that a reasonable number of plaques (ca. 50- 200) will form on the plates during the latent period. The dilution factor in going from the FGT (first growth tube) to SGT (second growth tube) is chosen on the basis of the expected increase in plaques at the end of the rise period so that later platings of SGT samples will also yield countable numbers. As mentioned in the bacteriophage section, it is recommended to perform a preliminary one-step experiment to estimate the possible length of the latent period and burst size. Then repeat the experiment with more frequent sampling with adjustments to the dilution factors to gain more precision. Sampling times may need to be adjusted for each phage-host system.<br /><br /><strong>In summary:</strong> <br /><br />1. Determine total phage input (Phage<sub>input</sub>) and total cyanobacteria input (Cyano<sub>input</sub>). <br /><br />2. At T = 0 determine the titer of unadsorbed phages (Free Phage<sub>T=0</sub>) and total infective centers (TIC<sub>T=0</sub> = initial total infected cells). <br /><br />3. During the latent period and rise, determine the titer of total infective centers (TIC = total infected cells + any phages released). <br /><br />4. At the end determine the titer of total progeny (TIC<sub>final</sub>). <br />Calculations. <br />Adsorbed phages = Phage<sub>input</sub> - Free Phage<sub>T=0</sub> <br />Percent adsorption = Adsorbed phages/ Phage<sub>input</sub> x 100 <br />Average MOI = Adsorbed phages/Cyano<sub>input</sub> <br />Burst size = (TIC<sub>final</sub>/TIC<sub>T=0</sub>)

[STEPS]
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TITLE: Double-fixation prior to ChIPs
AUTHORS: Jacob G Kirkland, Mary Bergwell, JinYoung Park

[DESCRIPTION]
This protocol is for the double-fixation of chromatin regulators for chromatin precipitation. The double-fixation method can improve signal-to-noise ratios of chromatin regulator ChIPs, especially for transiently bound proteins or those found in large protein complexes.

[STEPS]
SECTION: Fresh DSG
1. Place DSG at room temperature for approximately 30 minutes before weighing it out. 
SECTION: Fresh DSG
2. Measure DSG and dissolve in DMSO accordingly (using approximately 80 ul of solution per sample)
 
     Number of Samples      DSG (mg)      Vol. DMSO (ul)        2      13      160         4      25      320         6      38      480         12      72      960   



 
 


SECTION: Isolate and count single cells
3. Harvest the cells by first washing with PBS

SECTION: Isolate and count single cells
4. Incubate cells in trypsin-EDTA 0.25% until dissociated

SECTION: Isolate and count single cells
5. Quench trypsin with media + FBS and move cells to a 15ml conical. Spin down at 300g for 4 minutes
SECTION: Isolate and count single cells
6. Resuspend cells in PBS and count
SECTION: Isolate and count single cells
7. Move 30e6 cells to a new tube and bring the volume up to 10mls with PBS and spin down at 300g for 4 minutes
SECTION: Isolate and count single cells
8. Resuspend cells in 10mls fresh PBS
SECTION: DSG fixation (First)
9. Add 80 ul DSG stock to each sample in 10 ml of PBS and incubate for 30 minutes at room temperature with rocking. 
Note: After incubation with DSG, cells may adhere to the walls of pipette tips, so following
this incubation, it is recommended that tips be coated with 1% BSA/PBS
SECTION: DSG fixation (First)
10. Pellet samples by spinning at 1500 x g for 10 minutes at 4C
SECTION: DSG fixation (First)
11. Carefully aspirate the supernatant, taking care not to disturb the pellet.
SECTION: DSG fixation (First)
12. Resuspend each sample in 10 ml of CiA Fix Buffer
SECTION: Formaldehyde Fixation (Second)
13. Add 667 ul of 16% Formaldehyde to the sample dropwise and incubate for exactly 10 minutes at room temperature using rotation

SECTION: Formaldehyde Fixation (Second)
14. Stop cross-linking by adding 555 ul of 2.5M glycine.
SECTION: Formaldehyde Fixation (Second)
15. Incubate samples on ice for 5 minutes
SECTION: Formaldehyde Fixation (Second)
16. Pellet samples by spinning at 1500 x g for 10 minutes at 4°C

SECTION: Formaldehyde Fixation (Second)
17. Aspirate supernatant taking care not to disturb the pellet
SECTION: Formaldehyde Fixation (Second)
18. Wash fixed cells with 10ml PBS

SECTION: Formaldehyde Fixation (Second)
19. Pellet samples by spinning at 1000 x g for 5 minutes at 4C and aspirate supernatant. 
SECTION: Formaldehyde Fixation (Second)
20. Samples can be stored at -80°C (snap freeze) or can begin processing for chromatin immunoprecipitation
TITLE: EVALUATION OF TEST COMPOUNDS EFFICACY IN REDUCTION OF NEUROLOGICAL DEFICITS IN THE RAT CHRONIC CEREBROVASCULAR INSUFFICIENCY MODEL (CVI)
AUTHORS: Denis Kurkin, Dmitry Bakulin, Dmitry Verkholyak, Nikolay Kovalev, Evgeny Morkovin, Ivan Tyurenkov

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to provide detailed procedures for evaluating the effectiveness of test compounds (such as small molecules) in promoting brain ischemia rehabilitation in a rat model of chronic cerebrovascular insufficiency (by 50% stenosis of the common carotid arteries). The protocol compiled into pdf file. It contains the design of the experiment, including the description of the modeling of chronic cerebrovascular insufficiency and suitable methods for assessing the severity of pathology (assessment of sensory, motor and cognitive deficits).</div></div>

[STEPS]
TITLE: Human Brain Vascular Pericytes (HBVP) Fixation and Staining 
AUTHORS: Daniel Manrique-Castano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is suitable for the fixation and staining of cultured Human Brain Vascular Pericytes (HBVP).</div></div>

[STEPS]
?. [Cell fixation]
When cultured pericytes have reached the desired confluence on Poly-D-Lysine or Matrigel-coated glass coverslips, they can be fixed and prepared for staining. Before starting cell manipulation, warm 2% Paraformaldehyde (PFA) at   for .
37 °C
This procedure is unsuitable to perform RNA or protein extraction.
?. [Cell fixation]
To avoid cell damage by the abrupt change between the culture media’s and the fixation solution’s osmolarity, add  of 2% PFA during . After,  suction the medium and incubate the cells with   of 2% PFA during  at .
500 µl
500 µl
0 Room temperature
When adding substrates, pipette on the lateral walls of each wheel and not directly on to of the cells to avoid cell detachment or damage.
?. [Cell fixation]
Under the microscope, verify cell adherence to the glass coverslips
?. [Cell fixation]
Aspirate PFA, and Rinse three times with DPBS to remove fixative residuals.
?. [Cell fixation]
Keep cells in   DPBS at  until staining.
1 mL
4 °C
?. [Cell staining]
Recover cells from  and aspirate the DPBS.
4 °C
?. [Cell staining]
To permeabilize and block for unspecific staining, incubate the cells in  of Blocking-Permeabilization buffer (see materials) for  at
500 µl
0 Room temperature
?. [Cell staining]
Aspirate the Blocking-permeabilization solution, and incubate the cells in primary antibody buffer containing diluted primary antibodies  at .
4 °C
?. [Cell staining]
When incubation time is finished, wash the cells 3 times with PBS,  each.
?. [Cell staining]
Suction PBS, and incubate the cells with secondary antibody buffer containing diluted secondary antibodies  at .
4 Room temperature
?. [Cell staining]
Subsequently,  wash the cells 3 times with PBS,  each.
?. [Cell staining]
Finally, carefully detach the glass coverslip from the wheel plate using forceps, and place the cell surface facing a glass slide containing a small drop of  mounting media.
?. [Cell staining]
Left the mounting dry at  , and store the samples at  for further imaging.
0 Room temperature
4 °C
TITLE: Metadata extraction Zeiss CZI, before conversion to JPEG/TIFF (MacOS)
AUTHORS: scott john

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Extract metadata from Zeiss CZI files before conversion to TIFF/JPEG, using macOS.</div><div class = "text-block">Require "Fiji" and legacy Java 6 runtime for macOS 10.13</div></div>

[STEPS]
?. Cheat Sheet for those using Zeiss CZI files and need to extract metadata before saving/converting file to TIFF/JPEGOn a Mac- with that caveat:  MAC: Download Fijifrom https://imagej.net/Fiji/Downloads  (This also has windows version- care read caution file)MAC: You will also need Javaolder version; legacy Java 6 runtime for macOS 10.13https://support.apple.com/kb/DL1572?locale=en_USInstall Java then Fiji  Fijishould automatically update.  Open czi file.  Fijishould open window "Bio-Formats Import Options”  Under metadata viewing- check on:Display metadata,Display OME-XML metadata, Display ROIsClick “OK”  This opens three new windows:  1) Original metadata:- file name  2) OME Metadata:- file name  3) An image file:- file name.  Select window- Original metadata  Got to menu File: Save As  Exports the meta data as csv formatted file.   Can then save the image files as TIFF or JPEG or…Notes;Originally tested on CZI with no stacks.Work in progress
TITLE: Human Liver Sample Collection Protocol -- University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr, Steve Johnson

[DESCRIPTION]
Collection protocol obtained from the attached BioNet Specimen Procurement Agreement provided by the UMN CTSI Biorepository and Laboratory Services (BLS). 

  



[STEPS]
SECTION: Preparation
1. Patient Identification: As soon as a patient is scheduled, the research team will email bionet@umn.edu a completed Specimen Procurement Request Form.
SECTION: Preparation
2. Patient Consent: Researcher consents. The original signed consent form will be placed in the patient chart and scanned into Epic. BioNet will verify consent in Epic. If the consent is not scanned in EPIC before the procedure, the researcher will provide BioNet a copy direct (e-mail or hard copy).
SECTION: Preparation
3. Collection Supplies: All supplies will be delivered to the BioNet office at least one day before the procedure and appropriately labeled with researcher name and CTSI Project #.

Collection Kit Components:
 PBS (Study team)
 Media: DMEM + antibiotics (Study team)
 Media tubes (x3 per collection) (Study team)
 10% Formalin (Bionet)
 FFPE cassette x 5 (Bionet)
 Specimen container (Bionet)
 OCT compound (Bionet)
 OCT mold x 4 (Bionet)
 OCT quick freeze box (Bionet)
 Liquid nitrogen vapor (Bionet)
 Cryovials x 3 (Bionet)
 Collection kit containing 10 ml EDTA blood tubes x 2 (Bionet)
SECTION: Procurement and Processing of Liver Samples
5. Core 1:
Surgeon or OR staff will place core 1 in 10% formalin.
BLS procurement will pick up specimen and request Bionet Research Specimen Order.
BLS will tape a labeled FFPE cassette to the top of the specimen container.
BLS procurement will submit specimen to BLS Histology for FFPE processing. Standard fixation period of 24-72 hours. H&E and trichrome stain requested.
SECTION: Procurement and Processing of Liver Samples
4. 2 liver biopsy cores will be collected by surgeon.
The location of the tissue collection will be recorded by the surgeon.
BLS will be paged by OR for immediate specimen pickup.
SECTION: Procurement and Processing of Liver Samples
6. Core 2:
Surgeon or OR staff will place core 2 in specimen container and page BLS.
BLS procurement will pick up specimen immediately and request Bionet Research Specimen Order. Short transit/processing time is critical for this specimen.
Prepare OCT quick freeze box by adding liquid nitrogen. This must be done after the core is collected
 because the liquid nitrogen vapor will dissipate quickly.
Place core 2 in one OCT mold.
Ensure mold is labeled.
Add OCT compound into mold containing core. Ensure no bubbles surround the tissue.
After OCT sample is frozen, remove and transfer to -BOC freezer storage.
SECTION: Procurement and Processing of Liver Samples
7. If liver is collected as a wedge:
One wedge will be removed in the OR and placed in PBS.
Split wedge in half and treat as core 1 and core 2, processing according to same instructions.
TITLE: TMR labeling of LRRK proteins
AUTHORS: David Snead, Mariusz  Matyszewski

[DESCRIPTION]
Protocol for non-specific TMR labeling of LRRK1 and LRRK2 RCKW proteins.

Protocol developed by David Snead and adapted by Mariusz Matyszewski for protocols.io.

Written as used in Snead, Matyszewski, Dickey et al. 2022.

[STEPS]
1. Typical reaction volume is 40 µL .
Add dye in 1:1 ratio to about 20 micromolar (µM) .

 

2. Incubate at Room temperature for 60 min 

3. Remove excess dye by a buffer exchange through a Micro Bio-Spin P-6 desalting column (Bio-Rad). 
Make sure to equilibrate the column with the LRRK2 Storage buffer beforehand. Follow directions included with the column.
3.1. Repeat the exchange one more time to further remove the excess dye.
4. Quantify protein concentration and label efficiency using a NanoDrop or equivalent method.
TITLE: Media Prep: 500mL of 1x PBST
AUTHORS: Melanie Palma Avila, George  Testo

[DESCRIPTION]
PBST is commonly used as a wash solution for Western blot membranes and microtiter plate wells in ELISA assays. The Phosphate Buffered Saline with Tween® 20 is an optimal formulation of pH stabilizers, salts and detergents designed to effectively remove excess material from membranes and microtiter plate wells without disrupting the antigen/antibody binding reaction. By maintaining the proper buffering environment, unbound components can be washed away without suppressing antigen-antibody binding interactions, thereby reducing nonspecific background and increasing the specific signal.

[STEPS]
1. Perform a 1:10 dilution of 10x PBST using a sterile media bottle of 1L volume or greater (if necessary).
2. Place cap directly on top of sterile media bottle (DO NOT tighten cap).
3. Autoclave sterile media with 1x PBST: Liquid cycle: media (30 mins)
4. Retrieve autoclaved 1x PBST & fully cap sterile media bottle for storage.
1.1. Combine 450 mL DI H2O and 50 mL 10x PBST into sterile media bottle.
TITLE: Unilateral intranigral AAV alpha synuclein mouse model
AUTHORS: Pranay Srivastava, Waijiao Cai, Xiqun Chen

[DESCRIPTION]
This protocol details methods for the unilateral intranigral AAV alpha-synuclein injection

[STEPS]
1. Clean all equipment and surgical tools with 70% ethanol.
Turn on the heating pad and cover it with a paper towel.
2. Clean and prepare injector needle.
Rinse 3x with 70% ethanol.
Rinse 3x with saline.

3. Animals: adult mice.
Coordinators: from lambda AP +0.09 cm, ML+0.12 cm, DV -0.44 cm
Injection rate: 12 µl/hour, total 2 µl in 10 min.
4. Anesthesia: IP animals with Avertin solution
15 ml/10 g body weight. After approximately 3-5 minutes animal should be completely anesthetized. Verify this by observing no response to tail pinch.  
5. Fill the glass micropipette needle with virus solution (20 µl) by drawing the syringe plunger slowly. You can see a line between mineral oil and the solution. Adjust the rate to 12 µl/hour, volume to 2 µl. Pre-run the micro pump until you see a drop of liquid in the needle tip. Resume the volume to 2 µl.
6. Make a midline scalp incision and identify both the bregma and lambda coronal sutures.  Place animal on platform in prone position and place head in ear bars (ear bars should be aligned with the area just anterior to external ear). Place snout in incisor bar and tighten loosely.  Equilibrate bregma and lambda by measuring height of each using the stereotaxic arm to lower cannula until it contacts the exposed skull surface. A difference of less than 0.02 cm is acceptable. Adjust by raising or lowering snout height using incisor bar (be careful not to tighten snout holder since it will force wrong head angle).
7. From lambda dial in the following coordinates: AP: +0.09 cm, ML: +0.12 cm, lower needle tip to exposed dura and dial in: DV: -0.44 cm.
8. Infuse AAV alpha synuclein, at a rate of 12 µl/hour for 10 min (total 2 µL). Wait 2 minute and slowly withdraw needle 1 minutes after the end of the infusion
9. Suture scalp and place animal in incubator for recovery from anesthesia.
TITLE: BD FACS Aria II Start-up and Calibration
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol provides step by step instructions for performing single-cell sorting by using BD FACSAria II machine and BD FACSDiva software in preparation for single-cell sorting.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Data-Independent Acquisition (DIA) Data Processing using Spectronaut/directDIA (Biognosys): Secretome Analysis
AUTHORS: Joanna Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Proteolytic peptide measurement of secretome samples using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer for peptide/protein identification and quantification by processing DIA data using Spectronaut software.

[STEPS]
1. Search the DIA data with Spectronaut using the spectral library-free directDIA algorithm against a Human UniProt-SwissProt proteome database (https://www.uniprot.org/proteomes), applying the settings described in Table 1.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 1">
<div>
<div>
<p>Endo F1 cleaves Asparagine-linked high mannose or hybrid oligosaccharides. It cleaves between the two N-acetylglucosamine residues in the diacetylchitobiose core of the oligosaccharide, generating a truncated sugar molecule with one N-acetylglucosamine residue remaining on the asparagine. In contrast, PNGase F removes the oligosaccharide intact.</p>
</div>
</div>
</div>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is a parody of an existing scientific journal article and is intended to showcase the features available on the protocols.io platform in order for VERVE Net members to develope future protocols. While the viral metagenomic analysis on Cabbage Patch Kids is impossible to perform, due to the real-world non-existant nature of Cabbage Patch kids, the sourced article is <em>Metagenomic analysis of viruses associated with field-grown and retail lettuce identifies human and animal viruses </em>written by Tiong Gim Aw, Samantha Wengert, and Joan B. Rose (included in the authors section)and the "Material and methods" section has been adapted from using actual lettuce data to a metagenomic protocol for Cabbage Patch Kids.

[BEFORE_START]
It is advisable to obtain all necessary materials prior to obtaining field Cabbage Patch Kids. Cabbage Patch Kids mutate quickly from field removal and must be maintained in an adequate environment to prevent a syndrome what has recently been described as <em>Ravage Patch Kids.</em>

[STEPS]
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