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

[DESCRIPTION]
This protocol provides a method for assigning whole metagenome taxonomy using MetaPhlAn and MEGAN toolkits. Based on the methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Perl scripts and other supplementary information available at : <br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<strong>Required Software:</strong><br />
<ul>
<li>MetaPhlAn v1.7.7</li>
<li>prinseq-lite-0.20.3</li>
<li>NCBI's BLAST + v 2.2.0</li>
<li>MEGAN v5.5.3</li>
</ul>
<strong><br />Relevant Files</strong><br />Output:<br />
<ul>
<li>MetaPhlAn/skinmet_metaphlan_merged_output_genera.txt</li>
<li>MEGAN/megan_sk.txt</li>
<li>MEGAN/megan_genera_bacteria.txt</li>
<li>MEGAN/megan_genera_eukaryotes.txt</li>
<li>MEGAN/eukaryote_counts.txt</li>
<li>MEGAN/megan_genera-viruses.txt</li>
</ul>
Perl scripts: parse_megan_output.pl<br />R scripts: <a href="https://www.protocols.io/view/Script-R8-Plotting-Bacterial-Taxonomy-from-MetaPhl-ejbbcin" target="_blank">R8</a>, <a href="https://www.protocols.io/view/Script-R9-Plotting-Microbial-Taxonomy-from-MEGAN-ejdbci6" target="_blank">R9</a>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>March, 2012, based on RNAqueous May 29, 2008 protocol revision C, phenol/chloroform protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5438.full), ethanol precipitation protocol (http://cshprotocols.cshlp.org/content/2010/6/pdb.prot5440.full)</p>

[GUIDELINES]
<p>To avoid possible RNA degradation, try to work quickly in all steps.</p>

[STEPS]
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TITLE: Building a molecule for docking using PubChem and YASARA
AUTHORS: Chris Berndsen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The database </span><a href="https://pubchem.ncbi.nlm.nih.gov/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Pubchem</span></a><span> is a good resource for finding the structure of small, organic molecules as well as information on the structure, function, and effects of these small molecules. This information can be useful for finding information on how a drug might function or the structure of the drug itself. Using the SMILES information from a PubChem, it is possible to extract the structure for building the small molecule in the modeling program YASARA. This 3-D structure is then the ligand for docking studies with a target protein or for other computational analyses.</span></div></div>

[STEPS]
?. [Find the structure of your molecule]
Navigate to PubChem
?. [Find the structure of your molecule]
Search for the molecule name
The molecule will like not be a protein, but a small molecule like glucose or maltodextrin. Searching for proteins is not useful.
?. [Find the structure of your molecule]
Select an entry from the results and check to make sure that the molecule matches expectations.
?. [Find the structure of your molecule]
Record the PubChem CID (found in the Title/Summary section at top) as a note in this step.
?. [Find the structure of your molecule]
In section 3.1.4, copy the canonical SMILES code. Record it as a note on this step
?. [Building the molecule in YASARA]
Open YASARA
?. [Building the molecule in YASARA]
Go to Edit>Build>Molecule from SMILES string
?. [Building the molecule in YASARA]
Paste the SMILES string in the box and press OK
?. [Building the molecule in YASARA]
The resulting molecule will be built, however it may contain unusual geometry.
?. [Building the molecule in YASARA]
Right click on SMILES in the Scene Content table and select Name to rename the molecule. Record the name as a note on this step.
?. [Building the molecule in YASARA]
Save the molecule as a .PDB file. File>Save As>PDB file. Then save the file and upload to OSFName the file asligandname_smiles.pdbRecord the file location as a note on this step.
TITLE: TDP-43 RNA aptamer staining to detect pathological TDP-43 in FFPE human tissue, as described in Spence and Waldron et al., 2024 (Acta Neuropathologica): A SOP and tick-sheet. v2.
AUTHORS: Fergal M Waldron, Holly Spence, Jenna Gregory

[DESCRIPTION]
Here we provide a SOP to outline the correct procedures for performing Immunohistochemistry (IHC) to detect pathological TDP-43 in FFPE-preserved human tissue using the TDP-43 RNA Aptamer, as described in Spence and Waldron et al., 2024 published in Acta Neuropathologica (DOI: 10.1007/s00401-024-02705-1).

Users with access to Sequenza immunostaining racks and histological facilities (with fume hood) should be able to carry out all steps over two days.

This protocol uses the TDP-43APT sequence published in Zacco et al., 2022. The sequence is: CGGUGUUGCU with a  3' Biotin-TEG modification, purified using HPLC, scale: 1.0 µM synthesis.

 
References for citation of this method

Please cite both of these below if using this method:

The citation, Spence and Waldron et al., 2024, for the first publication for the development, modification and employment of theTDP-43 RNA aptamer to stain human tissue published in Acta Neuropathologica.

RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS.
Holly Spence*, Fergal M. Waldron*, Rebecca S. Saleeb, Anna-Leigh Brown, Olivia M. Rifai, Martina Gilodi, Fiona Read, Kristine, Roberts, Gillian Milne, Debbie Wilkinson, Judi O’Shaughnessy, Annalisa Pastore, Pietro Fratta, Neil Shneider, Gian Gaetano Tartaglia, Elsa Zacco, Mathew H. Horrocks, Jenna M. Gregory‡. Acta Neuropathologica 2024 Mar 5;147(1):50. DOI: 10.1007/s00401-024-02705-1. *equal contributions, ‡corresponding author.

The citation, Waldron and Spence et al., 2024, for this SOP published on protocols.io is:

TDP-43 RNA aptamer staining to detect pathological TDP-43 in FFPE human tissue, as described in Spence and Waldron et al., 2024 (Acta Neuropathologica): A SOP and tick-sheet. v2.
Fergal M. Waldron*, Holly Spence*, Jenna M. Gregory‡. protocols.io 2024; 
DOI: dx.doi.org/10.17504/protocols.io.eq2lyjo4mlx9/v2. *equal contributions, ‡corresponding author.     


[BEFORE_START]
Please see appendix for materials required to carry out this protocol.

[GUIDELINES]
This protocol is designed for users with access to Sequenza immunostaining racks and histological facilities (with fume hood) should be able to carry out all steps over two days.

[STEPS]
TITLE: Synapse Quantification with Puncta Analyzer
AUTHORS: Justin T Savage

[DESCRIPTION]
Protocol for installing Puncta Analyzer and using it to analyze synapse images.   

[STEPS]
SECTION: Install Puncta Analyzer
1. Go to https://github.com/toddstavish/puncta-analyzer and follow the following instructions for installing Puncta Analyzer
SECTION: Install Puncta Analyzer
2. Install JDK
SECTION: Install Puncta Analyzer
3. Install maven
SECTION: Install Puncta Analyzer
4. Fork the puncta analyzer repo
SECTION: Install Puncta Analyzer
5. Execute 'mvn compile' in project directory (where the pom.xml is located)
SECTION: Install Puncta Analyzer
6. Create a 'PunctaAnalyzer' directory in the your ImageJ plugins directory
SECTION: Install Puncta Analyzer
7. Copy all of the class files (bytecode) in target/classes to plugins/PunctaAnalyzer
SECTION: Running Puncta Analzyer
8. Z-project images if necessary
SECTION: Running Puncta Analzyer
9. Open an image in FIJI and use Ctrl+A to select all or select a specific region of interest
SECTION: Running Puncta Analzyer
10. Run Puncta Analyzer using Analyze>Puncta Analyzer
SECTION: Running Puncta Analzyer
11. Select Red channel and Green channel boxes to analyze those channels. Also select background subtraction for the red and green channels. Click set results file and save results and select a folder to save to.
SECTION: Running Puncta Analzyer
12. Click OK with the default 50 pixel rolling ball radius for subtract background
SECTION: Running Puncta Analzyer
13. Threshold the red channel using the slider
SECTION: Running Puncta Analzyer
14. Click OK with the default 50 pixel rolling ball radius for subtract background
SECTION: Running Puncta Analzyer
15. Threshold the red channel using the slider
SECTION: Running Puncta Analzyer
16. Enter any desired restrictions on the analyze particles function such as a minimum pixel size.
SECTION: Running Puncta Analzyer
17. Click yes to save result to file
SECTION: Running Puncta Analzyer
18. Close all windows and proceed to the next image
TITLE: Patient satisfaction survey at the multidisciplinary knee osteoarthritis clinic
AUTHORS: Rebeca Dias, Fabricio Loures, Henrique Chamarelli, João Henrique Reis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To evaluate the satisfaction of patients undergoing conservative multidisciplinary treatment for osteoarthritis of the knee at Hopstial Universitário Pedro Ernesto.</div></div>

[STEPS]
?. To survey de medical records of all patients who treated at the multidisciplinary knee osteoarthritis outpatient clinic at Hospital Universitário Pedro Ernesto.
?. Make contact and explain the content and importance of the research to improve the quality of assistance.
?. Conduct the interview with eight questions for patients who agree to participate.
?. Compile the data.
TITLE: BAF_Protocol_004 LC-MS(/MS) nLC EASY LC1200 and Orbitrap Exploris 480
AUTHORS: nesf

[DESCRIPTION]
This protocol gives a basic set up with LC (nLC 1200) and Instrument (Thermo Exploris 480) conditions for running proteomics samples with low to moderate complexity. This is what we use and can be adapted by shortening/lengthening gradient or changing MS parameters to fit the sample. We also use this when testing instrument performance with a standard HeLa digest.

[GUIDELINES]
These are our general settings and can be used as a starting point for most any proteomics experiment. Specific adjustments need to be made to meet specific samples or type of data required.

[STEPS]
SECTION: Prepare samples for injection
1. Suspend digested peptides with 20-40 uL of 0.1% FA. 
SECTION: Prepare samples for injection
2. Vortex, microfuge for 15 minutes at max speed, and take off 10uL into autosampler vial (do not touch bottom of tube). Use gel loading tip to transfer the volume to autosampler vial to prevent carrying any unsoluble material.
SECTION: Prepare samples for injection
3. Make sure there is no air in the bottom of the vial, carefully add the vial into the autosampler of the nLC 1200 system.
SECTION: nLC parameters- 2 h for complex samples
5. Sample pickup: 5 uL (loop size 20 uL) with a flow of 10 uL/min.

SECTION: Full scan parameters
13. Scan 375-1500 m/z, resolution 120K, time 60ms, normalized AGC 300%, RF lens 40%, 1 microscan
SECTION: General Instrument (Exploris 480)
12. Spray Voltage 1700V, Ion Transfer Tube 275C, All gases 0.
SECTION: Prepare samples for injection
4. In the sequence setup view of the Xcalibur software include each sample in a row filling with: file name, sample ID, folder directory to save results, directory for the acquisition method, vial position and injection volume. Normally use 10-50% of sample.
SECTION: nLC parameters- 2 h for complex samples
6. Sample loading: 20 uL, max. pressure 500 bar, no flow defined.
SECTION: nLC parameters- 2 h for complex samples
7. Gradient:
Flow = 300 nl/min
0 min 2%B; 3 min 2% B; 3-105 min 2-25% B; 105-125 25-40% B;  125-126 min 40-95%B; 126-131 min 95%B.

SECTION: nLC parameters- 2 h for complex samples
8. Pre-column equilibration: 10 uL, max. pressure 500 bar, no flow defined.
SECTION: nLC parameters- 2 h for complex samples
9. Analytical column equilibration: 8 uL, max. pressure 500 bar, no flow defined.
SECTION: nLC parameters- 2 h for complex samples
10. Autosampler wash: standard wash with 100 uL of flush volume.
SECTION: nLC parameters- 2 h for complex samples
11. EASY-spray: column heating on, column temperature: 40°C.
SECTION: MS/MS parameters
14. Min intensity 1E4, Charge +2 to +7, Dynamic exclusion - 1 time, 20s, 10ppm, isotopes, 10 MS2 scans, isolation 2 m/z, resolution 30K, HCD NCE 30%, first mass 100, time 60ms, normalized AGC 30%, 1 microscan.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Background
Physical activity reduces the risk of noncommunicable diseases and is therefore an
essential component of a healthy lifestyle. Regular engagement in physical activity can
produce immediate and long term health benefits. However, physical activity levels are
not as high as might be expected. For example, according to the global World Health
Organization (WHO) 2017 statistics, more than 80% of the world's adolescents are
insufficiently physically active. In response to this problem, physical activity programs
have become popular, with step counts commonly used to measure program
performance. Analysing step count data and the statistical modeling of this data is
therefore important for evaluating individual and program performance.This study
reviews the statistical methods that used to model and evaluate physical activity
programs, using step counts.

Methods
Adhering to PRISMA guidelines,this review systematically searched for relevant journal
articles which were published between January 2000 and August 2017 in any of three
databases (PubMed, PsycINFO and Web of Science). Only the journal articles which
used a statistical model in analysing step counts for a healthy sample of participants,
enrolled in an intervention involving physical exercise or a physical activity program,
were included in this study. In these programs the activities considered were natural
elements of everyday life rather than special activity interventions.

Results
This systematic review was able to identify 78 unique articles describing statistical
models for analysing step counts obtained through physical activity programs. General
linear models and generalized linear models were the most popular methods used
followed by multilevel models, while structural equation modeling was only used for
measuring the personal and psychological factors related to step counts. Surprisingly no
use was made of time series analysis for analysing step count data. The review also
suggested several strategies for the personalisation of physical activity programs.

Conclusions
Overall, it appears that the physical activity levels of people involved in such programs
vary across individuals depending on psychosocial, demographic, weather and climatic
factors. Statistical models can provide a better understanding of the impact of these
factors, allowing for the provision of more personalised physical activity programs,
which are expected to produce better immediate and long-term outcomes for
participants. It is hoped that this review will identify the statistical methods which are
most suitable for this purpose.

[STEPS]
SECTION: Define the review question
?. 
SECTION: Search
?. 
SECTION: Key words for the search
?. 
SECTION: Inclusion and exclusion criteria
?. 
SECTION: Selection Process and Data Extraction
?. 
SECTION: PRISMA flow diagram
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SECTION: Quality assessment - Risk of Bias Assessment
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SECTION: Results
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SECTION: Guideline
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TITLE: ONT DNA Barcoding Fungal Amplicons w/ MinION &amp; Flongle
AUTHORS: Stephen       Douglas Russell

[DESCRIPTION]
This collection of protocols outline a working methodology to DNA barcode fungal specimens. This process will work for dried tissue, fresh tissue, or with DNA template that has already gone through an alternative extraction protocol. It takes you from computer/software setup through DNA extraction, amplification, library preparation, and sequencing It also includes primary and secondary data analysis. Take your fungal samples from specimens to GenBank accessions with this protocol!

This particular workflow is designed for use with Flongle 10.4.1 flowcells and V14 Ligation chemistry.


[STEPS]
TITLE: Primary cortical neuronal culture
AUTHORS: Xiqun Chen, Qing Ye

[DESCRIPTION]
Primary cortical neurons were prepared from C57BL/6J mice embryonic day 17. The dissected cortical tissue was digested, triturated, and centrifuged. Cells were plated onto poly (L-lysine)-coated 24-well plates at 106 cells per well and cultured in NB-A with 2% B27 (Invitrogen, USA). After 24 h in culture, 5µg/ml cytarabine was added to inhibit the growth of glial cells in the medium and then changed to the original medium 48 h later. Neurons were cultured for 5 days and ready for experiments.

[STEPS]
1. For Primary cortical neuronal culture  - Use C57BL/6J mice at embryonic day 17 
2. Anesthetized pregnant mice (1% sodium pentobarbital, 80mg/kg), dissect their embryos and collect the cortex. 
(Separate and remove the soft membrane and blood vessels, rinse the cerebral cortex in PBS, and use the ophthalmic scissor to cut pieces of the cortex)
3. Collect the cortices in PBS  in a 50 ml tube on ice
(The 50 ml tube contains 30 ml of PBS) on ice 
4. Transfer the cortices to 15 ml tubes containing 1.5 ml trypsin–EDTA (0.25%) and incubate it at 37 °C for 15 min Dissociate the cortices by triturating with a 10 mL serological pipette 10 – 15 times
6. Triturate the cell suspension 10 times with a 1ml pipette
7. Coat 24 well plate with   (PLL)  
Place the cover glass at the bottom of the 24-well plate and add 400ul/well of PLL coating ( 0.01%) for 24h and incubate at 37 °C  
Wash 3 times with PBS after 1440 min 
8. Seed the cells onto PLL coated 24-well plates at a cell density of 106 cells/well containing specialized    supplemented with 2%  
Incubate for 1440 min at 37 °C 

9. Post 24 h, add 2µg/well of cytarabine (Stock - 5µg/ml) to the culture to inhibit the glial cell growth.
10. 2880 min later remove the medium completely  
Add 400µl/well of NB-A supplemented with 2% B27 
11. Transduction with BRAF (Optional)

After 5 days of culture, these cells are ready for further experiment
12. After 5 days, the neurons were transduced with BRAFV600E, or BRAFWT, or vector lentivirus with 8 μg/ml polybrene (Sigma–Aldrich, USA) for 24 h.
13. The cells were cultured in NB-A for 120 h and used for subsequent experiments.
5. Centrifuge the dissociated cortices (1500 rpm, 5 min) and resuspend the pellet in 10ml   medium supplemented with 10%
TITLE: Deposition of matrix using an M5 TM sprayer for high resolution MALDI analysis
AUTHORS: Martin Dufresne, Angela  Kruse, Jamie Allen, Danielle Gutierrez, Jeff Spraggins

[DESCRIPTION]
This protocol describes the use of an HTX M5 TM sprayer to deposit small molecule matrices onto tissue sections for high resolution MALDI imaging mass spectrometry (IMS). Spraying conditions will vary depending on matrix, and this protocol focuses on matrices for lipid IMS such as DMACA and DHB. 

[STEPS]
SECTION: Autofluorescence Scan
1. Remove slides from freezer and place in desiccator for 30 min 

SECTION: Autofluorescence Scan
2. Collect autofluorescence scan using a Zeiss AxioScan Z1: Pre-IMS Autofluorescence Microscopy
SECTION: TM Sprayer Setup
3. Set nitrogen flow to 8 psi. turn on TM sprayer. 
SECTION: TM Sprayer Setup
4. Open HTX software
SECTION: TM Sprayer Setup
5. Set nozzle temperature to 60C, and heated stage temperature (if applicable) to 45C.
SECTION: TM Sprayer Setup
6. Clean sample loop with 10mL of methanol with sprayer set to "load". Switch to "spray" and allow to spray for 2 minutes.
SECTION: TM Sprayer Setup
7. Make matrix solution of DMACA or DHB at a concentration of 12mg/mL in a solution of 60% THF and 40% acetone containing 5uL/mL DMF. Sonicate solution for 10 minutes.
SECTION: TM Sprayer Setup
8. Switch sprayer to "load" and load sample loop with 2 mL of matrix solution using a syringe.
SECTION: TM Sprayer Setup
9. Switch sprayer to "spray" and allow to spray for ~30 seconds. Check that matrix is spraying by placing a glass slide under the nozzle. 
SECTION: TM Sprayer Setup
10. Tape slide onto heated stage, and adjust the spray area in the HTX software. 
SECTION: TM Sprayer Setup
11. Set the method in the HTX software:
1350 mm/min nozzle velocity
1.5 mm track spacing
0.05 mL/min flow rate
CC pattern
4 passes
40 mm nozzle height
No drying time
SECTION: TM Sprayer Setup
12. Ensure that the method is highlighted. Press start under the Cycle tab. Click Continue. Wait for purge delay and confirm that the nozzle is moving
SECTION: TM Sprayer Setup
13. When matrix deposition is finished, remove the slides and store them in a slide box. Press "valve load confirm" in the software.
SECTION: Cleanup
14. Set TM sprayer nozzle and stage temperatures to 30C
SECTION: Cleanup
15. With sprayer set to "load", load loop with 10 mL each of acetonitrile followed by methanol. Set sprayer to "spray" and allow to flow for ~5 minutes. 
SECTION: Cleanup
16. Spray nozzle tip with methanol, taking care not to touch the tip. 
SECTION: Cleanup
17. Clean the stage with methanol. Do not spray solvents directly onto the stage. 
SECTION: Cleanup
18. Replace the bench liner in front of the instruments
SECTION: Cleanup
19. Dispose of syringes in biohazard container
SECTION: Cleanup
20. Shut down HTX software and close the computer. Turn off the TM sprayer.
SECTION: Cleanup
21. Shut off the nitrogen flow.
TITLE: C. elegans bleaching solution preparation
AUTHORS: Cristian Riccio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The </span><span style = "font-style:italic;">C. elegans</span><span> bleaching solution is used for several purposes: </span></div><div class = "text-block"><span>- destroying and removing bacterial or fungal cells from a contaminated </span><span style = "font-style:italic;">C. elegans</span><span> population. This is possible because </span><span style = "font-style:italic;">C. elegans</span><span> eggs are resistant to the bleaching used in this protocol but most fungi and bacteria are not.</span></div><div class = "text-block"><span>- synchronising a </span><span style = "font-style:italic;">C. elegans</span><span> population consisting of worms at different developmental stages. This is possible because eggs are resistant to the bleaching used in this protocol but hatched worms (larvae and adults) are not.</span></div></div>

[STEPS]
?. Add 42 ml DEPC water to a 50 ml Falcon tube.
?. Add 3 ml 10 M NaOH
?. Add 5 ml sodium hypochlorite solution
?. See relevant protocol for details but 6 ml of bleaching solution for 5 minutes on a vortex in 14 ml tubes should be sufficient to dissolve worms and spare eggs.
?. The bleaching solution can be kept at room temperature for one month.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Este protocolo describe los pasos para la recolección de hongos descomponedores de madera. Este protocolo fue elaborado bajo la guía de Daniela Torres (Fundación Fungi, http://www.ffungi.org) e información recolectada online.</p>
<p> </p>

[GUIDELINES]
<p>Se recomienda realizar la búsqueda en sectores de alta humedad o bosques con presencia de árboles muertos (caídos, talados o incluso quemados). No obstante, los hongos descomponedores de madera pueden encontrarse en árboles vivos.</p>
<p>La recolección puede tomar tejido del cuerpo fructífero del hongo o solamente su micelio, el cual típicamente es de color blanco para hongos de este tipo. La desventaja de esta última técnica es que dificulta la descripción, diferenciación e identificación de la especie.</p>
<p> </p>

[STEPS]
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