eamag/protocols-with-steps huggingface dataset:
TITLE: bright field big patch swarming imaging
AUTHORS: Serena Ding

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For imaging swarming behaviour of 40 young adult C. elegans on a big food patch on agar using the Phoenix multi-worm tracker system. Worms are synchronised by bleaching and refeeding for 72 hours, and then 40 young adult hermaphrodites are transferred by glass pipette onto a 35 mm regular NGM plate for imaging for 20 hours at 25 fps.</div></div>

[STEPS]
?. [Imaging plate preparation (Day -7)]
A separate batch of imaging plates is poured exactly seven days before each imaging day and stored at 4°C.
Imaging plates are 35 mm Petri dishes containing 3.5 mL low peptone (0.013% Difco Bacto) NGMagar (2% Bio/Agar, BioGene) to limit bacteria growth.
?. [Bleach synchronising worms (Day -7 to -4)]
Bleach synchronise gravid hermaphrodites. Leave on rotator at 20 °C until use.
?. [Re-feed worms (Day -3, PM)]
Re-feed starved L1 worms onto 3-4 plates using a glass pipette. Incubate at 20 °C.
Culture plates are 55 mm Petri dishes containing 15 mL low peptone 0.013% Difco Bacto) NGMagar (2% Bio/Agar, BioGene), and seeded with OP50.
?. [Imaging (Day 0)]
Forty animals are transferred by a glass pipette onto the imaging plate in a small drop of M9, away from the bacteria lawn.
?. [Imaging (Day 0)]
Imaging commences immediately following animal transfer in a liquid drop, on a custom-built six-camera rig (the Phoenix) equipped with Dalsa Genie cameras (G2-GM10-T2041). Twenty-hour recordings with red illumination (630 nm LED illumination, CCS Inc.) are taken at 25 Hz using Gecko software (v2.0.3.1), whilst the rig maintain theimaging plates at 20 °C throughout the recording durations.
?. [Image data processing]
Images are segmented in real time by the Gecko software.
?. [Imaging (Day 0)]
Wash animals off of culture plates with M9 and collect in a 15 mL Falcon tube, wash in M9 twice, and aspire as much supernatent as possible after the last wash.
?. [Image data processing]
The recordings are manually truncated post-acquisition to retain aggregation and swarming dynamics only.
The start time is defined as the moment when the liquid dries and the all the worms crawl out from the initial location of the drop, and the end time is when the food is depleted and worms disperse with increased crawling speed.
?. [Seeding imaging plate (Day -1, PM)]
The center of an imaging plate is seeded with a single 75 μL spot of cold diluted OP50 (OD=0.38) and allowed toinoculate overnight at room temperature before being used for imaging the next day.
TITLE: Characterization of osmoregulated periplasmic glucans with high resolution LCMS
AUTHORS: lcmsmethods  , Benjamin C. Orsburn, Colten Eberhard, Allison Daitsch, Namandje N. Bumpus

[DESCRIPTION]
Characterization of osmoregulated periplasmic glucans with high resolution LCMS

[STEPS]
1.   
 
  
These are optimized methods for the characterization of osmoregulated periplasmic glucans as described in Allison Daitsch et al., 2022. 
Briefly: All analysis was performed on a Dionex UHPLC and Q Exactive quadrupole Orbitrap system (Thermo Fisher). Two micrograms of each reaction and unreacted input was injected directly onto a HyperSil Gold C-18 2.1mm x 150mm reversed phase chromatography column. Analytes were separated using an increasing gradient that consisted of 0.1% formic acid in LCMS grade water as buffer B and 0.1% formic acid in LCMS grade acetonitrile as buffer B. Due to the hydrophilic nature of glucans, the gradient began with a 2-minute acquisition at 100% buffer A with a rapid ramp to 100% buffer B by 15 minutes before returning to baseline conditions for the remainder of the 20 minute experiment. The Q Exactive was operated in positive ionization mode using a data dependent acquisition method. An MS1 scan was acquired at 140,000 resolution with a scan range of 150 to 1500 m/z. The three most abundant ions from each MS1 scan were isolated for fragmentation using a three-step collision energy of 10, 30 and 100 and the fragment scans were obtained using 15,000 resolution. Ions with unassigned charge states or more than 3 charges were excluded from fragmentation. To prevent repeat fragmentation any ion within 5 ppm mass deviation of the selected ion was excluded from additional fragmentation for 30 seconds. All Thermo .RAW instrument files have been uploaded to the MASSIVE public repository under accession MSV000089142 The vendor .RAW files and processed results can be accessed during the review process using the following link: ftp://MSV000089142@massive.ucsd.eduand reviewer password EstG725

A second method with the suffix "high_mass" is a recommended method for the characterization of OPGs of higher m/z charge ratio. Both methods can be utilized in both positive ionization mode as provided here, or in negative ionization mode by changing the polarity of the Q Exactive acquisition method. Low mass OPGs of interest for this study were identified solely with the positivetop32142021  method. Higher mass and negative polarity ionization may be necessary for the characterization of alternate OPG specis.
TITLE: ssDNA2.0: Ligation mix I
AUTHORS: Matthias Meyer, Sarah Nagel, Anna Schmidt

[DESCRIPTION]
Protocol for the preparation of Ligation mix I for the first adapter ligation in automated single-stranded DNA library preparation using the ssDNA2.0 method (Gansauge et al. 2020). 

References
Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols, 15, 2279-2300.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p class="p1"><span class="s1">Hurwitz, Bonnie L., and Matthew B. Sullivan. "The Pacific Ocean Virome (POV): a marine viral metagenomic dataset and associated protein clusters for quantitative viral ecology." <em>PLoS One</em> 8.2 (2013): e57355.</span></p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: 16S_PCR_Sequence_Analysis
AUTHORS: Kenneth Acosta

[DESCRIPTION]
Protocol to generate a consensus 16S rRNA gene sequence from forward and reverse sequences.  

[STEPS]
SECTION: Adding U515 sequence feature
1. Open Serial Cloner. 
 

SECTION: Adding U515 sequence feature
2. Go to “Features” → “Manage Features”.
SECTION: Adding U515 sequence feature
3. Add new collection. Name “16S rRNA Gene Conserved Regions”.
SECTION: Adding U515 sequence feature
4. Add new feature. Name “U515”. Add sequence “GTGCCAGCAGCCGCGGTAA”.
SECTION: Adding U515 sequence feature
5. Check “Scan For This Feature” box. Click Ok.
SECTION: Generating consensus 16S rNA gene sequence
6. Upload 16S rRNA gene forward and reverse sequences for the sample.
SECTION: Generating consensus 16S rNA gene sequence
7. Go to “Function” → “Align Two Sequences”.
SECTION: Generating consensus 16S rNA gene sequence
8. Check “anti-parallel” box for reverse sequence.
SECTION: Generating consensus 16S rNA gene sequence
9. Click “Local Align”.
SECTION: Generating consensus 16S rNA gene sequence
10. Click “Build Consensus”. New dialog box will appear.
SECTION: Generating consensus 16S rNA gene sequence
11. Select “Unsaved Consensus” window then click “Features” → “Scan Sequence”. “U515” feature should be highlighted in consensus sequence.
SECTION: Generating consensus 16S rNA gene sequence
12. Click “Features” tab in the “Unsaved Consensus” window.
SECTION: Generating consensus 16S rNA gene sequence
13. The nucleotide positions of the “U515” marker will be given. Select the “from” box in top right corner of window and type in the nucleotide position that is -385 nt from the beginning of the “U515” marker. Select the “to” box in top right corner of window and type in the nucleotide position that is +216 nt from the end of the “U515” marker. A 620 bp consensus sequence should be generated.
SECTION: Generating consensus 16S rNA gene sequence
14. To check for quality of consensus sequence select “Align Two Sequences” window and look for differences in base calls between forward and reverse sequences within the consensus sequence. Sometimes there will be a mismatch or gap between forward and reverse sequences. To assist in making the right base call at a certain nucleotide position use FinchTV to view chromatograms and determine the right call using the higher-quality base call.
 

SECTION: Generating consensus 16S rNA gene sequence
15. Save consensus 16S rRNA gene sequence.
SECTION: Generating consensus 16S rNA gene sequence
16. Annotate consensus 16S rRNA gene sequence by searching a database (e.g. NCBI) or using a 16S rRNA gene classifier (e.g. RDP classifier).
TITLE: RSV whole genome sequencing using ONT 
AUTHORS: Kseniia Komissarova

[DESCRIPTION]
Respiratory Syncytial Virus (RSV) is a leading cause of hospitalization due to acute lower respiratory infection in infants and young children. Monoclonal antibodies palivizumab is available option for treating RSV limited to select populations in high-resource settings. There is no vaccine to prevent RSV to the moment. Fortunately, several vaccine candidates are now in the human testing phase targeting young children, older adults and pregnant women, and an effective safe vaccine is likely to be available in the near future.

For vaccine and therapy development RSV surveillance is essential. Using whole-genome sequencing data helps ensure the vaccine has the most up-to-date coverage and more-effective global reach. Through genomic surveillance we can track the spread of variants, monitor changes to the genetic code of the virus. Finally, this data is used to better understand how virus might impact public health.

Here we provide the protocol for genome sequencing of RSV A using Oxford Nanopore Technology platform. The four overlapping genome fragments together comprise of the full RSV genome encompassing all viral genes, yet lacking the far 3′ and 5′ genome termini.

[STEPS]
1. Order primers according to the list from your local manufacturer
 

3. Briefly vortex and centrifuge defrosted reagents before use.
Prepare four RT-qPCR reactions for every RNA sample you need to amplify for sequencing. 
Use one pair of primers above (forward and reverse) per reaction
Prepare the PCR reaction mixture following the specifications below:

 

2. Dilute primers to the concentration of 10mM. Aliquote and freese.
3. Extract viral RNA from the samples using QIAamp Viral RNA Kits (QIAGEN)
5. Assess quantity and quality of amplicons using capillary or gel DNA electrophoresis.
Lenght of the products are: 4665, 4086, 3802, 4431 b.p.
4. Use next cycling conditions for your thermocycler:
1. 55 °C for 10 min 
2. 98 °C for 2 min, 
3. 40 cycles of 98 °C for 10 s, 61 °C for 10 s and 72 °C for 3 min
6. For each sample combine four PCR reactions in one volume (100 mcl summary)
7. Prepare library with amplified samples using NEBNext® ARTIC SARS-CoV-2 Companion Kit for Oxford Nanopore Technologies® 

Follow protocol E7660 on protocols.io beginning with 12 step.
https://www.protocols.io/view/nebnext-artic-sars-cov-2-companion-kit-oxford-nano-dm6gpwd78lzp/v4?step=12
TITLE: In vitro kinase activity
AUTHORS: Xinbo Wang, Pietro De Camilli

[DESCRIPTION]
This protocol details methods for the in vitro kinase activity testing of purified LRRK2.

[STEPS]
SECTION: In vitro kinase activity
1. Set up the reaction mixtures with 1x kinase buffer (diluted from 10x kinase buffer), 200 nanomolar (nM) LRRK2 or 8 µg Rab8 protein, for the reaction group add 1 millimolar (mM) ATP, and for the control add H2O instead, the total volume we used is 40 µL.
SECTION: In vitro kinase activity
2. Incubate samples for 120 min at 30 °C.
SECTION: In vitro kinase activity
3. Quench reactions through addition of SDS sample loading buffer and heat at 95 °C for 10 min.
SECTION: In vitro kinase activity
4. Resolve samples by SDS/PAGE or Phos-tag SDS/PAGE.
SECTION: In vitro kinase activity
5. Detect proteins by coomassie blue staining or western blot using antibodies of Rab8 and LRRK2 phospho-specific (pT1357), respectively.
TITLE: Freezing Fresh Tissue
AUTHORS: Jamie Allen, Maya Brewer, Elizabeth Neumann, Danielle Gutierrez, Mark deCaestecker, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">To describe the procedure for freezing fresh tissue procured from the Cooperative Human Tissue Network.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">Frozen tissue should have minimal structural damage from freezing.</div></div>

[STEPS]
?. [Freezing and Documentation]
Place a layer of dry ice in to box and add enough isopentane to make a slurry.
?. [Freezing and Documentation]
Label embedding molds and add a small amount of CMC to each one (barely cover the bottom).
?. [Freezing and Documentation]
Remove sample cup from ice and document on sample sheet the information on lid.
?. [Freezing and Documentation]
Remove sample with forceps. Measure and document the length, width and height.  Document size and conditions (attachment)
?. [Freezing and Documentation]
Place metal weigh boat on top of slurry and let cool.  Add tissue and cover box with lid.
?. [Freezing and Documentation]
Allow tissue to completely freeze.
?. [Freezing and Documentation]
Place embedding mold with CMC on top of the slurry.
?. [Freezing and Documentation]
Check the mold in the slurry to see if it is starting to freeze.  Add tissue when the CMC becomes only “tacky” on top.  Orient the tissue so that the side that is to be cut is face down.
?. [Freezing and Documentation]
Slowly add more CMC on top of the tissue until it is covered.  Keep the lid closed over the slurry as much as possible.
?. [Freezing and Documentation]
?. [Freezing and Documentation]
When completely frozen, wrap in foil and label sample number with sharpie.
?. [Freezing and Documentation]
Place all samples from one patient in a labeled box and place in the.
-80 °C
?. [Freezing and Documentation]
Place disposable equipment in the Biohazard trash.  Wipe all forceps, rulers, bottles with appropriate cleansing wipes.
?. [Formalin Fixed Tissue]
If fixing in formalin, prepare 1 large jar of 10% formalin.
?. [Formalin Fixed Tissue]
During step 4, carefully slice a small section of tissue (preferably having both cortex and medulla).  Do this in a way that you create two "bookends."  The section for fixation should be no thicker than a nickel.
?. [Formalin Fixed Tissue]
Carefully place the piece of tissue into an embedding cassette.
?. [Formalin Fixed Tissue]
Allow tissue to “fix” overnight.
?. [Formalin Fixed Tissue]
Move tissue cassettes into 70% ethanol.
?. [Formalin Fixed Tissue]
Tissue is then paraffin embedded by the pathology Core.
TITLE: Staining Spores for Conidia Counting
AUTHORS: IAC, Sara de Oliveira Olimpio, Rebecca Ogioni

[DESCRIPTION]
Methylene blue has different functions depending on its concentration. Its use allows the visualization of dead tissues and bacteria, for example. As a solution, this dye allows the identification of microorganisms in general. For practical use in this matter, the morphological identification of the microorganism is obtained from the visualization of its conidia with the specific dye.

[STEPS]
SECTION: Procedure
1. Add sample in 1,5 mL tube;

SECTION: Procedure
2. Add 100 uL of methylene blue dye;
SECTION: Procedure
3. Homogenize by vortexing for 10 seconds and wait 2 minutes;
SECTION: Procedure
4. Centrifuge for 1 minute at 16.000g;
SECTION: Procedure
5. Pipette 50 uL into the Neubauer chamber or microscope slide and cover with a cover slip;
SECTION: Procedure
6. Evaluate under an optical microscope;
SECTION: Materials
8. Tween® 80;
SECTION: Materials
9. Methylene blue; 
SECTION: Materials
10. Distilled water.
13. Pipette out 20 uL of Tween® 80;
SECTION: Soluction Preparation (100 mL)
12. Weigh 0.7g of methylene blue;
14. Fill volume to 100 mL with distilled water.
SECTION: Soluction Preparation (100 mL)
11. In a becker add 80 mL of distilled water;
SECTION: References
15. Pham, Tuan Anh et al. “Production of Blastospore of Entomopathogenic Beauveria bassiana in a Submerged Batch Culture.” Mycobiology vol. 37,3 (2009): 218-24. doi:10.4489/MYCO.2009.37.3.218
SECTION: Procedure
7. Store at 4ºC overnight before to use.
SECTION: References
16. Oktari, A., et al. "The bacterial endospore stain on Schaeffer Fulton using variation of methylene blue solution." Journal of Physics: Conference Series. Vol. 812. No. 1. IOP Publishing, 2017.
TITLE: Nutil Data Integration
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol describes Nutil data integration from segmentation and registration of mouse brain data.

[STEPS]
SECTION: Nutil Data Quantification
1. Open Nutil and find operation tab.
 
SECTION: Nutil Data Quantification
2. Find the 3 folders you created at the beginning of this workflow and populated during the workflow and populate them into their corresponding list above. (segmentation is your input folder, brain atlas map folder is your atlas folder, output folder is output and should be empty still XML or JSON anchoring file is the QuickN JSON file that you created earlier – you can also use visualign json file it doesn’t matter the anchor is set in quickNII and nonlinear adjustments are made in reference to it. Object color Is black because that’s what the staining of our segmentations look like if it were a different color we would need to look for another object color code it corresponds to during the segmentation step.
 
SECTION: Nutil Data Quantification
3.      
Navigate to advanced settings in the row labeled Global Pixel Scale/Unit, set it to the resolution of your classifier, converted to area per pixel (um^2/px). This can be calculated by squaring the resolution value in the QuPath thresholder (given in um/px-length). For example, if using brightfield images scanned at Van Andel (usually scanned at .503 um/px) and using the “Very High” resolution in QuPath (scaled by factor of 2), the resolution you would put into Nutil would be 1.02 um^2/px ( or (1.01um)^2/px). Set Pixel Scale Unit to um^2

 
It is mainly important to have this completely accurate if your analysis contains variables that take area into account (e.g. +ve objects/um^2). It is not as crucial if only looking at percent area occupied as ratios will not be affected by scale conversions. More info on this can be found in the Nutil docs FAQs, item 9.6: https://nutil.readthedocs.io/en/latest/FAQ.html 
SECTION: Nutil Data Quantification
4. Also, under advance settings change custom masking to yes and select the color of the hemisphere of interest i.e. white to view hemisphere masked in white mask. Also select the folder location of the mask. Repeat the color selection with black to view the other hemisphere masked by black.
TITLE: PLASMA- 01 - Plasma separation from human whole blood
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Massimiliano Legnaro, Emanuela Rasini, Marco Ferrari, Franca Marino

[STEPS]
?. Put whole blood (at least ) into a 5 mL tube.
1.5 mL
?. Centrifuge at 
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Centrifuge: 1400 34, 5 min
?. Carefully collect the supernatant (plasma). Do not touch the red pellet on the bottom.
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≤ 85% of plasma should be retrieved to avoid contamination by the underlying cell pellet.
?. Store plasma at -80°C until use. Defrost only once.
?. EXPECTED RESULTS
Expected plasma yield is approximately 50% of whole blood volume.
TITLE: Collection and shipment of specimen for single-nuclei RNA sequencing (snRNA-Seq)
AUTHORS: Laura Robinson, Susan Sheehan, Gaven Garland, Ron Korstanje

[DESCRIPTION]
These samples are part of the JAX-Sen project in the SenNet Consortium. Here we provide details on specimen collection and shipment to the Robson laboratory at The Jackson Laboratory for Genomic Medicine (JAX-GM) in Farmington, CT for its processing for single-nuclei RNA-sequencing (snRNA-seq).

[STEPS]
SECTION: Reagents and Materials
1. 2mL Cryovials/ screw-cap tubes
ice cold 1X PBS
Kimwipes
Petri dishes
Liquid Nitrogen
Dry ice
Tweezers (clean, sterile)
SECTION: Quality Key Points:
2. The tissue specimen should be kept at 4 degrees Celsius and RNase-free until snap-freezing and maintained at -80 degrees C or on dry ice thereafter, until it is processed at JAX. 
It is crucial to not store the tissue specimen at RT to avoid freeze-thaw cycles, any cell death, and tissue and/or RNA degradation. 
SECTION: Procedure:
3. Timeline: The daily shipping deadline at Jax BH is 12:00 noon. So, harvest the tissues before and closer to noon.
SECTION: Procedure:
4. Collection/Harvest:
SECTION: Procedure:
4.1. Animal was euthanized via cervical dislocation. 
SECTION: Procedure:
4.2. Animal was pinned to a necropsy tray which remained on wet ice throughout the harvest.
SECTION: Procedure:
4.3.  Animal was perfused with 20ml cold PBS.
SECTION: Procedure:
4.4. Heart, pancreas, and kidney was collected and the specimen was collected and the specimen was rinsed with ice-cold 1x PBS in a Petri dish to remove blood and other debris.
SECTION: Procedure:
4.5. Pat the specimen dry using clean Kimwipes and freeze as quickly as possible after harvest. 
SECTION: Procedure:
4.6. To flash freeze (snap freeze), place the tissue in a cryo-tube and submerge the tube in liquid nitrogen or immerse deeply in dry ice.
SECTION: Procedure:
4.7. Wait at least 2-3 minutes for the tissue to completely freeze, and transfer the tube containing the tissue to -80 degrees C.
SECTION: Procedure:
4.8. Keep at -80 degrees Celsius thereafter, until shipping.
SECTION: Procedure:
5. Shipment:
SECTION: Procedure:
5.1. Place sample tubes in a plastic box (cardboard boxes insulate the samples from the cold ice) or in double Ziplock bags after checking that they are completely sealed.
SECTION: Procedure:
5.2. Ship the sample box on dry ice (O/N shipping) to:
The Jackson Laboratory for Genomic Medicine,
Farmington, CT, 06032
TITLE: 0.3M Sodium Cacodylate Buffer pH 7.4 Stock Solution Recipe
AUTHORS: Mark Ellisman, Marianne Martone, Thomas Deerinck

[DESCRIPTION]
Standard preparation protocol for stock 0.3M sodium cacodylate buffer. 

[GUIDELINES]
Cacodylate acid, sodium salt, trihydrate (MW = 214.03)
Vendor: Ted Pella
Product number: 18851

Calculation:

M (moles/liter) = g/ MW (g/mol) X V (liter)

0.3M = g/214.03 g/moles X 1.0L

g = M x V x MW

g = 0.3 moles/liter X 214.03 g/moles X 1.0 liter

g = 64.209 grams for 1.0 liter

For 3L = 3 x 64.209 = 192.627 grams

[STEPS]
1. Add 900 ml of DDH2O to 64.209 grams of sodium cacodylate in a large 1.0L or greater container, such as a capped glass bottle. Shake and wait until powder is fully dissolved.

2. Maximum HCL concentration is 12.18mole/liter (36-38%). Start with 1.3mL of 12.18 M HCL per 900 ml of sodium cacodylate dissolved solution. Make sure solution is mixed thoroughly. 

4. Top off to 1000ml with DDH2O.
5. Do a final pH check and keep stock solution refrigerated at 4C. 
3. Check pH. Keep adding 50-100μl of HCL, mix, and check pH again until pH 7.4 has been reached. (Approximately 1.4 ml of conc. HCL total).
TITLE: MSD + S + AS
AUTHORS: Eleanor Harman

[DESCRIPTION]
.

[STEPS]
SECTION: Media
1. MS Media: 4.4g/L 2.2g/500mL
Sucrose: 30g/L 15g/500mL
Sorbitol(5%): 50g/L 25g/500mL
Thymidine: 300mg/L 150mg/500mL
2,4D: 2mg/L 1mg/500mL
Phytagel(1.4%): 14g/L 7g/500mL
SECTION: Media
2. Add 80% final volume DI water to beaker
SECTION: Media
3. Add MS Media, Sucrose, Sorbitol, Thymidine, and 2,4D until fully dissolved
SECTION: Media
4. Adjust pH to 5.8

SECTION: Media
5. Add DI water until final volume
SECTION: Media
6. Autoclave on 20 minute liquid cycle