eamag/protocols-with-steps huggingface dataset:
TITLE: Measuring effects of down-regulated genes
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Synechocystis sp. PCC 6803 with integrated guide RNA culture</li><li style = "counter-reset:ol0;list-style-type:disc;">Synechocystis sp. PCC 6803 control</li><li style = "counter-reset:ol0;list-style-type:disc;">    inoculate 30 ml, grow for a few days</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Inducer </li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block"><span style = "font-weight:bold;">Start</span></div><div class = "text-block"><span>Dillute each culture to oD</span><span style = "vertical-align:sub;">750</span><span>=0,2</span></div><div class = "text-block">Add appropriate amount of antibiotics</div><div class = "text-block">Start with adding appropriate amount of Inducer </div><div class = "text-block"><span style = "font-weight:bold;">Following Days</span></div><div class = "text-block"><span>Measure oD</span><span style = "vertical-align:sub;">750 </span><span>of </span><span style = "font-style:italic;">Synechocystis sp</span><span>. PCC 6803 cultures</span></div><div class = "text-block"><span style = "font-weight:bold;">Day 3</span></div><div class = "text-block"><span>Measure oD</span><span style = "vertical-align:sub;">750</span><span> eventually last time (depends on how good your datas and your remaining culture is)</span></div><div class = "text-block">Centrifuge 5 ml of each culture</div><div class = "text-block">Resuspend pellet in pgtx for later use (storage: -80°C)</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<section>
<p><a href="http://bioinformatics.psb.ugent.be/webtools/trapid/" target="_blank">TRAPID</a> is an online tool for the fast, reliable and user-friendly analysis of <em>de novo</em> transcriptomes, developed and maintained by the CNB group at VIB-UGent Center for Plant Systems Biology.</p>
<p> </p>
</section>
<p>This protocol explains how to perform advanced functional and comparative analyses after a transcriptome dataset was functionally annotated using TRAPID. </p>

[BEFORE_START]
<p>In order to use TRAPID, make sure that:</p>
<ul>
<li>You are using a web browser with JavaScript and Adobe Flash enabled. Supported browsers: Safari, Chrome, Firefox (most recent versions).</li>
<li>Java is installed on your machine (version JRE 6.0 or higher). This is required to launch the JalView alignment editor and the Archeopteryx tree viewer.</li>
</ul>

[GUIDELINES]
<p>In this protocol, we work with an example dataset consisting in 25,392 transcript sequences from <em>Panicum hallii </em>(<a href="https://doi.org/10.1111/j.1365-313X.2012.04938.x" target="_blank">Meyer et al. 2012</a>). This dataset can be retrieved from TRAPID's FTP <a href="ftp://ftp.psb.ugent.be/pub/trapid/datasets/panicum" target="_blank">here</a>.</p>

[STEPS]
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TITLE: Coleta de dados actigráficos - ActTrust
AUTHORS: Daniel Vartanian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Esta coleção reúne os protocolos do processo de coleta de dados actigráficos do Grupo Interdisciplinar de Pesquisa em Sono (GIPSO). Esses protocolos foram desenhados para os actígrafos provenientes da companhia </span><a href="https://www.condorinst.com.br/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Condor Instruments</span></a><span>. Algumas das configurações mencionadas podem ser específicas desses aparelhos.</span></div><br/><div class = "text-block">Se você não é membro ou parceiro do GIPSO, você pode utilizar esta coleção como referência para o processamento de seus equipamentos. Neste caso, por favor, não envie os formulários mencionados nos protocolos relacionados ao processo.</div><br/><div class = "text-block"><span>Se você utilizar os protocolos do GIPSO em sua pesquisa, por favor considere citá-los na seção de materiais e métodos de sua estudo. Nós nos esforçamos bastante para elaborar e manter protocolos de acesso aberto à comunidade científica. Você pode encontrar a citação dos protocolos na aba</span><span style = "font-weight:bold;font-style:italic;"> </span><span style = "font-style:italic;">metadata</span><span>.</span></div><br/><div class = "text-block"><span>Consulte a aba </span><span style = "font-style:italic;">Guidelines </span><span>para visualizar o fluxograma do processo como um todo.</span></div><br/><div class = "text-block"><span>Para dúvidas ou sugestões, entre em contato pelo e-mail </span><a href="mailto:gipso@usp.br" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">gipso@usp.br</span></a><span> .</span></div></div>

[STEPS]
TITLE: Phylogenomic analysis of Xanthomonas
AUTHORS: David J Studholme

[DESCRIPTION]
This is a protocol for using PhaME to generate a phylogenomic tree from a set of Xanthomonas spp. genome sequences.

[STEPS]
9. Install PhaME software into a Conda environment called 'phame', following instructions on the software's GitHub page:
 
The PhaME software is described in this paper:
 

10. 
 

7. Set-up the reference genome sequence data
 

 

 

 

8. Set-up the working directory.

 

 


 
Optionally, at this point, we can delete the symbolic links for any genomes that we want to exclude from the final analysis. It is also an option to add any genome assemblies as .contig files (inf FASTA format).
 


3. Ensure that NCBI Datasets command line tools are installed and executable (or a symbolic link to the executable) is in the current directory.
 

4. Download the genome assemblies that will be included in the analysis:

 

 



 

 



 

 



1. Create a directory for downloaded genome sequence data:

 

2. Enter the directory for downloaded genome sequence data: 
 

6. Come back out of the directory for downloaded genome sequence data:

 

5. Rename the symbolic links to more informative names. We will use the rename_files.pl script to effect this.
 

 

 


 


11. Create file phame.ctl in current directory.

 





12. Execute PhaME:
 

 

13. The tree file can now be visualised using any tree-viewing software, for example, iTOL.
TITLE: 0.9% Saline Solution
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to prepare 0.9% Saline Solution. 0.9% Sterile Saline solution is used as an initial rinse for the brains of mice prior to perfusion. </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: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for measurement of carbon isotope descrimination during photosynthesis.</p>
<p> </p>
<p>See the following references for more background:</p>
<p>H. Griffiths (1993) <a href="https://link.springer.com/book/10.1007/978-94-011-1566-7" target="_blank" rel="noopener noreferrer">Photosynthesis and Production in a Changing Environment</a> pp 181-1927</p>
<p>Farquhar et al.  (1989) Annu. Rev. Plant Physiol. Plant Mol. Bioi. 1989. 40:503-37 </p>

[STEPS]
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TITLE: Visualign Transformation
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol describes visualign transformation.

[STEPS]
SECTION: Visualign Transformation
1. Open Visualign program from folder on the desktop. 
SECTION: Visualign Transformation
2. File > Open > select “QuickN JSON” you created in QuickNII.
SECTION: Visualign Transformation
3. Drag the opacity bar all the way to the right to where it says outline, and you will have all outlined regions displayed. This is the easiest format for transformation.
SECTION: Visualign Transformation
4. Align all regions properly.
SECTION: Visualign Transformation
4.1. Hover over an atlas region. 
SECTION: Visualign Transformation
4.2. Click the SPACE bar. 
SECTION: Visualign Transformation
4.3. Drag the cross symbol to the correct location.
SECTION: Visualign Transformation
4.4. Crosses can be moved at any point. They can also be deleted by hovering over them and clicking Delete on the keyboard.
 

SECTION: Visualign Transformation
5. Click the < and > arrows at the top right to navigate between sections. There is no need to “Store” these as in QuickNII. See a finished section below.
 
SECTION: Visualign Transformation
6. Once completed, click File > Save As > save “Visualign JSON” in the QVN folder. This can be opened again to continue aligning later.
SECTION: Visualign Transformation
7. To convert into a flat file, click File > Export, navigate to the Atlas folder and click “Select Folder”. This will export the files needed for Nutil.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Isolation of 40-200 µg of total RNA from <em>Synechocystis</em> sp. PCC 6803. Can be used for standard applications such as Northern Blot and qPCR.</p>

[BEFORE_START]
<p>PGTX solution (100 mL): 39.6 g phenol, 6.9 mL glycerol, 0.1 g hydroxyquinoline, 0.58 g EDTA, 0.8 g NaOAc, 9.5 g guanidine thiocyanate, 4.6 g guanidine hydrochloride</p>

[GUIDELINES]
<p>Keep RNA cold and in an RNase-free environment.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>Keeps cells always on ice &amp; all reagents must be ice-cold</p>
<p> </p>

[STEPS]
SECTION: Start a culture two days before transfection to get the culture full of amoebas cells
?. 
SECTION: Count the cells to reach 1.5E6 cells / condition
?. 
SECTION: Spin cells down at 2500g for 5'
?. 
SECTION: Wash cells with chilled 1xPBS
?. 
SECTION: Add 20ul of P3 buffer (Lonza) / condition
?. 
SECTION: Add 1-3 ug of a circular plasmid / condition
?. 
SECTION: Transfer cells + DNA into a well
?. 
SECTION: Insert into a Lonza machine and apply a code: EN-138
?. 
SECTION: Immediatelly add 80ul of Marine Broth medium (MB) to a well
?. 
SECTION: Transfer to 1ml of growth medium in 12-well plate (NUNC) and incubate overnight
?. 
SECTION: Check for positive cells 24h later using fluorescent microscopy
?.
TITLE: Biospecimen collection and processing 2.0
AUTHORS: John Herndon, Ryan Fields, Daniel Cui Zhou, Li Ding

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ABSTRACT</div><div class = "text-block">This is a description of the steps that the Washington University in St. Louis HTAN center has developed to collect surgical resection biospecimens, for molecular and genomic characterization. There are two separate protocols because the technique has evolved and better methods for tumor sampling have been developed. This is the second sample protocol and employs an innovative grid system for sampling.</div></div>

[STEPS]
?. Record time that the tumor sample arrived in the pathology gross lab
?. Examine the gross tumor sample with a Pathologist or Pathology Assistant
?. Once the tumor mass has been identified, photograph the tumor in situ
?. Request that the Pathologist remove a tumor piece that is ideally 2.0 cm2 x 0.5cm thick.
?. Request Pathologist orient the specimen by noting pertinent anatomical directions, i.e. anterior, posterior, lateral, medial
?. Photograph specimen with orientation markings, be sure there is no identifiable PHI, i.e. date.
?. Place specimen on cutting board and measuring with a ruler, cut the tumor sample in a grid style fashion to obtain anywhere from 2 to 9 pieces of equally sized subdivisions of tissue. Each subdivision should be 0.65 cm2.When squares are completely cut, photograph with a ruler to later annotate and subsequently measure the spatial relationships among the pieces.
?. For each grid square:turn the tissue square on it's side between the teeth of a pair of forceps and with a fresh scalpel blade begin slicing the tissue at an approximate thickness of 1mm.
?. Place the superior slice (A1) in to one well of a 24 well tissue culture dish with 10% Formalin, label appropriately
?. Place the next slice (A2)  in to a cryo mold, fill with OCT, and place on to a metal block surrounded by dry ice to freeze.Record the time.
?. Place the next slice (A3)  in to either DMEM or Flash freeze in liquid nitrogen. This will depend on the characterization assay to be performed using that slice.Record the time.
?. Place the last slice (A4) which is the inferior slice in to one well of a 24 well tissue culture dish with 10% formalin, label appropriately.
?. Repeat the above steps 8-12 for each tumor subdivision.
?. All the above steps assume a tumor slice that is uniform in size at about 2.0 cm2 x 0.5 cm. Tumors may vary in size and priorities will have to assessed in order to determine what samples can be taken and for what downstream processes can be done. Be prepared to improvise!
TITLE: Making no peptone NGM for imaging plates
AUTHORS: Bonnie Evans

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">C. elegans</span><span> is maintained in the laboratory on Nematode Growth Medium (NGM) agar seeded with bacteria. For imaging plates, NGM without bactopeptone is used to prevent the seeded bacteria growing too much, resulting in poor imaging.</span></div></div>

[STEPS]
?. [Pre-autoclave]
Book the autoclave via the notebook on top of the machine. It will take around 2 hours for a 500ml bottle and 2.5 hours for 1L or larger bottles.
?. [Pre-autoclave]
Take clean bottles and measuring cylinders from the glass kitchen - you will also need one bottle for the autoclave probe, which should be the size of the largest bottle you are autoclaving. Get sterile water from the media room.
?. [Pre-autoclave]
Measure out all the pre-autoclave reagents and add to the bottle. Use a new weighing boat and spatula for each reagent. Add water last and mix thoroughly.
?. [Pre-autoclave]
Add equivalent amount of water to the empty probe bottle.
?. [Pre-autoclave]
Make sure the lid of your media bottle is not screwed completely when placing it inside the autoclave machine. Label with autoclave tape (‘NGM agar Rm 5020’), and place over the lid to prevent it coming off in the autoclave.
?. [Autoclave]
Turn ON the autoclave and touch the screen
?. [Autoclave]
If 'Fill with water' is on the screen, fill up the autoclave with sterile water until it reaches the grill/'Water Level OK' appears on the screen.
?. [Autoclave]
Place the probe bottle inside the autoclave with the temperature probe inside.
?. [Autoclave]
Place the bottles in the autoclave and make sure that the cap is not screwed completely.
?. [Autoclave]
Check the waste flask is not too full
?. [Autoclave]
Close the door
?. [Autoclave]
(On the screen) LOCK --> START --> MEDIA 121X15 BALLAST --> Start
?. [Post-autoclave]
When autoclave is finished, UNLOCK the door
?. [Post-autoclave]
Remove the probe and media bottles using gloves (the bottles will be hot)
?. [Post-autoclave]
Leave the agar on a lab bench to cool to around 55°C, ie the bottle is cool enough to hold for a second with a gloved hand
?. [Post-autoclave]
Add the post autoclave reagents
?. [Post-autoclave]
Try not to shake the bottle too much while mixing to avoid air bubbles.
?. [Post-autoclave]
The agar needs to be warm to be poured without blocking the tubings, so try to pour as quickly as possible and if not poured immediately put the bottle on a waterbath set to 60C until being used.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">Increasing energy expenditure by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or induction of browning of white adipose tissue (WAT) is considered a promising strategy to treat/prevent obesity and related metabolic diseases</span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">. Whereas WAT is adapted to store energy as triglycerides, BAT produces heat (non-shivering thermogenesis). In brown adipocytes, the uncoupling protein-1 (UCP-1) regulates conversion of energy into heat by uncoupling ATP production from mitochondrial respiration</span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">. Also in WAT adaptive UCP-1 positive adipocytes (brown in white: brite or beige) can arise, predominantly in subcutaneous (s) WAT</span><span lang="EN-US" style="margin: 0px; line-height: 200%; font-family: 'Arial',sans-serif; font-size: 10pt;">. This browning of WAT is enhanced by exposure to cold.</span></p>

[GUIDELINES]
<p><span lang="EN-US" style="margin: 0px; line-height: 107%; font-family: 'Calibri',sans-serif; font-size: 11pt;">-Mouse needs to be out of cage, relaxed on a bench, a scalpel is used to cut of 1 mm of tail, the tail starts to bleed, glucose levels are measured on the second drop of blood.<br />-When the body temperature of the animals drop below 32°C, the experiment will be stopped (approved by the ethical committee of the KU Leuven).<br /></span></p>

[STEPS]
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TITLE: Environmental sampling using Moore swabs
AUTHORS: Dilip  Abraham, Nicola Elviss, Nicholas Feasey, Nick  Grassly, Jacob John, Gagandeep Kang, John  Scott Meschke, Venkata  Raghava Mohan, Satheesh Nair, Jonathan  Rigby, Rajan Srinivasan, Catherine  Troman, Christopher  Uzzell, Nicolette Zhou

[DESCRIPTION]
A protocol for the collection of environmental samples (sewage and wastewater) using Moore swabs and subsequent laboratory processing up to DNA extraction. This is part of a set of protocols for environmental surveillance of Salmonella Typhi. 

Bacteria in the wastewater are trapped in the Moore swab. Trapped bacteria are enriched during incubation in Universal Pre-Enrichment broth. The broth is then filtered under a vacuum to capture the bacteria on filter discs ready for DNA extraction. 

This protocol is adapted from the Moore swab protocol described in Liu et al (2021).

[BEFORE_START]
Ensure that you wear appropriate PPE during sampling (e.g. gloves, disposable lab coat, appropriate footwear,  facemask).

[GUIDELINES]
It is recommended that sample collection be done in the morning.

[STEPS]
SECTION: Sample Collection
1. Sterilise Moore swabs by autoclaving prior to taking them to the field site.
SECTION: Sample Collection
2. Immerse the swab in the sewage or wastewater channel and tie it to a stable support using the tailing thread of the swab.
SECTION: Sample Collection
3. Leave in place for 24-72 hours
SECTION: Sample Collection
4. Prepare 450ml of Universal Pre-Enrichment broth per sample, following the manufacturer's instructions
SECTION: Sample Collection
5. Cut the thread of the Moore swab and transfer the swab to an appropriately labelled container with 450ml of UPE broth
SECTION: Sample Collection
6. Place the container on ice and transport back to the lab within 4 hours of sample collection
SECTION: Processing the Moore Swab
7. Incubate the container with the Moore swab in UPE broth at 37 °C  for 21 - 24 hours

SECTION: Processing the Moore Swab
8. After incubation, swirl the mixture and pipet two 20ml aliquots from the enrichment broth into 50ml conical flasks


SECTION: Processing the Moore Swab
9. Assemble the membrane filtration unit according the manufacturer's instructions and attach to the vacuum pump
SECTION: Processing the Moore Swab
10. Using sterile tweezers, place a 47mm 0.45µm filter disc onto the filtration unit head and attach the filtration unit cup
SECTION: Processing the Moore Swab
11. Pipet the first 20ml sample into the filtration cup and turn on the vacuum to filter the sample through the disc
SECTION: Processing the Moore Swab
12. Once complete, cut the filter into several strips using sterile forceps then place the strips into an empty PowerBead tube. If possible, place the strips so that the "dirty" side faces the inside of the tube. 
SECTION: Processing the Moore Swab
13. Repeat steps 10 to 12 with the second 20ml sample.
SECTION: Processing the Moore Swab
14. Store samples at -20 °C  until extraction.

SECTION: Processing the Moore Swab
15. Thoroughly disinfect filtration cups, containers and conical flasks after use to avoid cross contamination of samples.
TITLE: 10Xv3.1 Genomics Sample Processing
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for the rapid generation of 3’ transcriptomic-NGS-ready- single-cell-libraries from pools of cells.</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's 1U01MH114812-01 and 1U19MH114830-01. 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: CALCIUM STAINING PROTOCOL
AUTHORS: Machlusil Husna, Kusworini Handono, Hidayat Sujuti, Aulanni'am, Ettie Rukmigarsari

[DESCRIPTION]
Neurodegeneration due to neurotoxicity is one of the phenomena in temporal lobe epilepsy. Experimentally, hippocampal excitotoxicity process can occur due to kainic acid exposure, especially in the CA3 area. Neuronal death, astrocyte reactivity and increased calcium also occur in hippocampal excitotoxicity, but few studies have investigated immediate effect after kainic acid exposure. The organotypic hippocampal slice culture (OHSC) is a useful model for studying the neurodegeneration process, but there are still many protocol differences. In this study, minor modifications were made in the OHSC protocol.

[STEPS]
SECTION: Calcium assay kit
1. We use Fluo-4 assay kit (calcium ab 228555) that provides a homogenous fluorescense-based assay for detecting the intracellular calcium mobilization
SECTION: Material supplied in kit
2. Fluo-4 AM 1 vial
10x F127 Plus 1 bottle (10 mL)
HHBS (Hank's Buffer with 20 mM Hepes) 1 bottle (100 mL)
SECTION: Reagent Preparation
3. Briefly centrifuge small vials at low speed prior to opening
Thaw all the kit components at room temperature before use
Fluo-4 AM stock solution : add 200 μL of DMSO into the vial of Fluo-4 AM and mix well
1x Assay buffer : make 1x assay buffer by adding 1 mL of 10x F127 plus into 9 mL of HHBS buffer and mix them well
Fluo-4 AM dye-loading solution : add 20 μL of Fluo-4 AM stock solution into 10 mL of 1x assay buffer and mix them well. This working solution is stable for at least 2 hours at room temperature
SECTION: Assay procedure
4. Equilibrate all materials and prepared reagents to room temperature just prior to use and gently agitate
Add 20 μL Fluo-4 AM dye-loading solution into the cell plate
Incubate the dye-loading plate in incubator with temperature 37oC fo 1 h
Prepare the plate with HHBS
Run the calcium flux assay by monitoring the fluorescense intensity at Ex/Em = 490/525 nm with CLSM