eamag/protocols-with-steps huggingface dataset:
TITLE: Quality guidance on the use of run controls for direct detection of poliovirus by nanopore sequencing
AUTHORS: Joyce Akello, Manasi Majumdar, Alex Shaw, Catherine Troman, Erika Bujaki, Javier Martin, Nick Grassly

[DESCRIPTION]
This document describes the laboratory process for the use of run controls (positive and negative controls) for performing direct detection of poliovirus by nanopore sequencing (DDNS assay). 


[STEPS]
TITLE: Transmission electron microscopy
AUTHORS: Sue Lin

[STEPS]
?. Double fixation: The specimen was first fixed with 2.5% glutaraldehyde in phosphate buffer (pH7.0) for more than 4 hours; washed three times in the phosphate buffer; then postfixed with 1% OsO4 in phosphate buffer (pH7.0) for 1 hour and washed three times in the phosphate buffer.
?. Dehydration: The specimen was first dehydrated by a graded series of ethanol (50%, 70%, 80%, 90%, 95% and 100%) for about 15 to 20 minutes at each step, transferred to absolute acetone for 20 minutes.
?. Infiltration: The specimen was placed in 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1 hour at room temperature, then transferred to 1:3 mixture of absolute acetone and the final resin mixture for 3 hours and to final Spurr resin mixture for overnight.
?. Embedding and ultrathin sectioning: Specimen was placed in capsules contained embedding medium and heated at 70℃ for about 9 hours.  Ultrathin sections (70 nm) were obtained and stained by uranyl acetate and alkalinelead citrate for 15 minutes respectively, and photographed by a Hitachi Model H-7650 transmission electron microscope (Tokyo, Japan).
TITLE: The Alliance Formation Puzzle in Contests with Capacity-Constraints: A Test using American Football Reception-Coverage Contest Data
AUTHORS: Justin Ehrlich, Yang-Ming Chang, Matthew Harmon, Shane Sanders

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>We utilize a contest-theoretic model to demonstrate a version of the alliance form puzzle that aligns with reception-coverage contests in American football.Namely, secondary defenders can opt for single-coverage (1 v 1 contest) or form a defensive alliance (double-coverage or 2 v 1 contest with exogenous intra-alliance prize division) when defending a given receiver.In our theoretical treatment, we find that defenses have a lower equilibrium success rate in preventing the receiver from “getting open” under double-coverage than under single-coverage in the absence of (sufficiently-binding) capacity constraints.We also find that this success rate paradox is a necessary condition for the alliance formation puzzle (i.e., loss of expected allied party payoffs under alliance).We then test the theoretical treatment by analyzing 8,508 plays of NCAA and NFL game data within a set of fixed effects, logistic regression models that control for receiver, level-of-play, and season-of-play.We find that equilibrium level of defensive success rises significantly (p-value < .01) and substantially (marginal effect of between 12.8 and 17 percentage points) when moving from single-coverage to double-coverage, </span><span style = "font-style:italic;">ceteris paribus</span><span>.There is strong evidence that the necessary condition for the alliance formation puzzle does not hold in this setting.We conclude that sufficiently-binding (physiological and training-based) capacity constraints eliminate the alliance formation puzzle in this setting (as was shown theoretically by Konrad and Kovenock, 2009).This empirical result suggests that other contest settings that regularly feature alliance (e.g., liquidity-constrained conflict) may not be puzzling.</span></div></div>

[STEPS]
?. Load football.do in Stata/SE 14.2 or newer. The latest version is located here: https://github.com/Syracuse-University-Sport-Analytics/Alliance-Formation-Puzzle-in-Professional-Football
?. Download football data.dta from https://dataverse.harvard.edu/dataset.xhtml?persistentId=doi:10.7910/DVN/ZIQQXO
?. Change line 2: use"C:\football data.dta", clear to point to the locatin that you saved football.do
?. Run football.do
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Extraction of high molecular weight DNA after Bolger et al.  </p>
<p> </p>
<p>Carried out for <em>eucalyptus grandis. </em></p>
<p>DNA fragments were ranging up to 270 kb on a Pulsfield Electrophoresis Gel </p>

[GUIDELINES]
<p>Modified from Section 1.3 of Supplementary materials from Anthony Bolger et al., The genome of the stress-tolerant wild tomato species <em>Solanum pennellii</em>, Nature genetics, Vol 46,1034-1038 (2014)  doi:10.1038/ng.3046</p>
<p> </p>
<p>Link: <a href="http://www.nature.com/ng/journal/v46/n9/full/ng.3046.html#supplementary" target="_blank">http://www.nature.com/ng/journal/v46/n9/full/ng.3046.html#supplementary</a></p>
<p> </p>
<p>When citing please make sure to mention the original Bolger et al. protocol as described above. </p>
<p> </p>
<p>Thank you very much Benjamin Schwessinger and Jasmine Saban for carrying out the extraction with me. </p>
<p> </p>
<p><strong>Reagents required: </strong></p>
<p> </p>
<p><strong>Extraction Buffer</strong> (pH 6.0):</p>
<p>1 M 2-methyl-2,4-pentanediol </p>
<p> </p>
<p>10 mM PIPES (1,4-Piperazinediethanesulfonic acid sodium salt)</p>
<p>10 mM MgCl2</p>
<p>4% (w/v) PVP-10</p>
<p>10 mM sodium metabisulfite</p>
<p>25 mM 2-mercaptoethanol</p>
<p>0.5% (w/v) sodium diethyldithiocarbamate</p>
<p>200 mM L-lysine</p>
<p>6 mM ethylene glycol tetraacetic acid (EGTA), </p>
<p> </p>
<p><strong>Nuclear Buffer</strong> (pH 7.0):</p>
<p>0.5 M 2-methly-2,4-pentanediol</p>
<p>10 mM PIPES</p>
<p>10 mM MgCl2</p>
<p>0.5% (v/v) Triton X-100</p>
<p>10 mM sodium metabisulfite</p>
<p>60 mM 2-mercaptoethanol</p>
<p>200 mM L-lysine</p>
<p>6 mM EGTA, pH 7.</p>
<p> </p>
<p><strong>TE Buffer</strong> (pH 7.0): </p>
<p>10 mM Tris</p>
<p>1 mM EDTA</p>
<p> </p>
<p><strong>Others solutions: </strong></p>
<p>Triton X-100</p>
<p>20% (w/v) SDS (Sodium dodecyl sulfate)</p>
<p>5 M sodium perchlorate</p>
<p>Phenol/Chloroform/Isoamylalcohol (25:24:1)</p>
<p>Chloroform/Isoamylalcohol (24:1)</p>
<p>100% Ethanol</p>
<p>70% Ethanol (fresh)</p>
<p>TE buffer </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>
<p> </p>

[STEPS]
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TITLE: Chlorophyll Extraction and Spectral Analysis 
AUTHORS: Victor Rodriguez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is designed to be able to extract  and analyze the concentration of chlorophyll within a sample of a given plant. The procedures of this protocol require using a spectroscope  to determine the approximate level a chlorophyll  within a given sample.</div></div>

[STEPS]
?. [Extraction of Chlorophyll ]
Weigh out  of sample subject and add it to a pestle.
0.25 g
?. [Extraction of Chlorophyll ]
Weigh out  of  and add it to a pestle.
0.13 g
?. [Extraction of Chlorophyll ]
Add  of to the pestle.
1 ml
?. [Extraction of Chlorophyll ]
Grind the entire mixture until the sample is consistent and theis completely dissolved into the sample paste.
?. [Extraction of Chlorophyll ]
Add the paste into a 40 ml beaker and wash out the pestle with   of  and empty it into the beaker.
1 ml
?. [Extraction of Chlorophyll ]
Add  of to the 40 ml beaker and mix the sample thoroughly, ensuring to press and remix the sample several times.
13 ml
?. [Extraction of Chlorophyll ]
Let the sample beaker stand for 15 minutes.
?. [Extraction of Chlorophyll ]
Filter the sample into a 20 ml test tube using a 20 micron retention filter paper.
?. [Extraction of Chlorophyll ]
Let the sample stand for a further 10 minutes.
?. [Sample Analyzation ]
Add  of the sample to two PCR tubes and label them according to your needed labeling system. the two tubes will serve as double tests for verification.
200 µl
?. [Sample Analyzation ]
Centrifuge the two samples to 1200 g to settle out any particulates for 1 minute.
?. [Sample Analyzation ]
From each PCR tube, transfer  of the sample from the top to another PCR Tube.
100 µl
?. [Sample Analyzation ]
Place each tube in the spectrometer.
?. [Sample Analyzation ]
Take to log base 10 of the absorbance percentage of the wavelength 647 nm and 664.5 nm and denote them as A647 and A664.
?. [Sample Analyzation ]
To calculate the concentration...CHLA=20.47A647 - 4.73A664CHLB= 12.63A664 - 2.52A647CHLTotal= CHLA + CHLBDivide each concentration by 1000, multiply by the acetone used, then divide by the sample mass to get the mg/g of sample
?. [Sample Analyzation ]
Confirm readings match to within an acceptable margin of error.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>To analyze a specific genome region using <span lang="EN-US">next-generation sequencing technologies</span>, the enrichment of DNA libraries with targeted capture methods has been standardized. For enrichment of mitochondrial genome, a previous study developed an original targeted capture method that use baits constructed from long-range polymerase chain reaction(PCR) amplicons, common laboratory reagents, and equipment. In this protocol, a new targeted capture method is presented, that of bacterial artificial chromosome (BAC) double capture (BDC), modifying the previous method, but using BAC libraries as baits for sequencing a relatively large gene.</p>

[GUIDELINES]
<p>This targeted enrichment was conducted following the modified protocol of Maricic et al., (2010) and the NimbleGen technical note “Double Capture: High Efficiency Sequence Capture of Small Targets for use in SeqCap EZ Library, Applications on 454 Sequencing Systems.” Please get the two protocols and check those in detail.</p>

[STEPS]
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TITLE: Optical coherence tomography angiography in age-related macular degeneration
AUTHORS: Miklos Resch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">All subjects underwent a comprehensive ophthalmic examination, including best-corrected visual acuity (VA) assessment (ETDRS Charts), slit lamp, and fundus examinations. After pupil dilation, OCT-A was performed, using the AngioVue OCT-A system (RTVue-XR Avanti, OptoVue, Fremont, CA, USA). Scans with the highest resolution were obtained in the central 3×3 mm area, centered on the foveola. The superficial capillary plexus was detected automaticaly between the internal limiting membrane (ILM) and the inner plexiform layer (IPL); and the deep capillary plexus between the IPL and the outer plexiform layer (OPL). Segmentation errors were manually controlled for each layer to avoid the potential artifacts due to the distortion of anatomy with nAMD.</div><div class = "text-block">Foveal thickness was measured in the central 1.0 mm area. Superficial and deep vessel density (VD) was evaluated in the whole image, in the central 1 mm area (fovea), and in the 3 mm ring-shaped parafoveal area. Superficial non-flow area and foveal avascular zone (FAZ) areas were measured, using the built-in AngioAnalytics software (Version ReVue 2018.0.0.18) OptoVue system with an automated segmentation algorithm. </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Leukemia is a malignant clonal disease of hematopoietic stem cells, also known as "blood cancer." Leukemia cells proliferate and accumulate in bone marrow and other hematopoietic tissues due to uncontrolled proliferation, differentiation disorders, and blocked apoptosis, and infiltrate other non-hematopoietic tissues and organs, while inhibiting normal hematopoietic function.</p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em>Asterionella formosa</em> is a freshwater pennate diatom that forms star-shaped colonies, whose mitochondrial DNA was recently sequenced <span style="font-size: 10.8333px;">[1]</span>. This diatom is of interest for genetic studies <span style="font-size: 10.8333px;">[2,3]</span> and for the study of photosynthesis <span style="font-size: 10.8333px;">[4,5].</span> However, extraction of genomic DNA (gDNA) from <em>A</em>. <em>formosa</em> is difficult because of its silica cell-wall and the presence of photosynthetic pigments that contaminate the DNA. Here, we present an optimised protocol for gDNA extraction from <em>A</em>. <em>formosa</em> that overcomes those two problems. We also assessed its efficacity in yielding high-purity gDNA compared to a standard protocol using hexadecyltrimethylammonium bromide (CTAB).</p>
<p> </p>
<p>References</p>
<p>[1] Villain A. et al. (2017) Mitochondr. DNA. 2 (1), 97-98.</p>
<p>[2] De Bruin A. et al. (2004) J. Phycol. 40, 823-830.</p>
<p>[3] van Den Wyngaert S. et al. (2015) Mol. Ecol. 24, 2955-2972.</p>
<p>[4] Boggetto N. et al. (2007) J. Phycol. 43, 1227-1235.</p>
<p>[5] Erales J. et al. (2008) J. Phycol. 44, 1455-1464.</p>

[STEPS]
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TITLE: Cell deposition and drug sensitivity assay
AUTHORS: Lev Tsypin, Aaron Turkewitz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to deposit several cells of </span><span style = "font-style:italic;">S. robusta</span><span> in droplets on a petri dish, allowing the experimenter to track the growth of approximately single cell isolates with high throughput. This was used to determine drug sensitivity.</span></div><div class = "text-block"><span>We found that in, liquid culture, </span><span style = "font-style:italic;">S. robusta</span><span> is sensitive to 10 ug/mL puromycin and 250 ug/mL glyphosate. One caveat to keep in mind is that </span><span style = "font-style:italic;">S. robusta</span><span> appears more tolerant to antibiotics when grown on agar plates, being insensitive to up to 20 ug/mL of puromycin, for example. We recommend selection in liquid culture.</span></div></div>

[STEPS]
?. [Measure density of floating cells in culture]
Grow 100 mL volume of cells in a 175 cm2 flask for three to four days, until there is a substantial number of healthy, floating cells
?. [Measure density of floating cells in culture]
Stand flask vertically and take a 15 mL volume of the floating culture (so as to not disturb the adherent cells) to measure cell density
?. [Measure density of floating cells in culture]
Measure cell density using a hemocytometer. The target concentration for distributing cells is 400-500 floating cells/mL, so dilute as necessary. The volume needed is 2 mL, including any drugs you want to add.
?. [Deposit cells in droplets on a petri dish]
b.	Take the 2 mL, containing the desired number of cells, in an Eppendorf tube.
?. [Deposit cells in droplets on a petri dish]
Centrifuge using a personal mini-centrifuge for two minutes. At this speed (which I believe is 6000 rpm), many cells remain in the supernatant. Those are the cells we want. Do not disturb any pellet that forms from the centrifugation.
?. [Deposit cells in droplets on a petri dish]
Using a Pipetteman, withdraw 40µl aliquots from near the top of the Eppendorf tube.  Deposit 40µl drops in a 6x8 array in a Petri dish.
?. [Count initial cells]
Record the approximate number of cells deposited in each drop.
?. [Count initial cells]
In our hands, this is ~4-10 cells per droplet on average
?. [Count initial cells]
Wrap petri dish with parafilm and place in incubator
?. [Assay]
After three days of treatment, count cell numbers/determine cell viability in each droplet
?. [Measure density of floating cells in culture]
Spin cells down at 6000xg to pellet them. Scrape and resuspend in 1 mL
TITLE: General DNA Extraction with Sigma Extract-N-Amp
AUTHORS: Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes general DNA Extraction with Sigma Extract-N-Amp.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Imaging single SYTOX Orange molecules on a PLL-coated cover glass
AUTHORS: Ezra Bruggeman

[DESCRIPTION]
This is a protocol for the preparation of a microscopy sample of single SYTOX Orange molecules on a PLL-coated cover glass. This protocol was used to generate the data shown in Figure 1a, 1b and 1c of the following publication:

Bruggeman et al., POLCAM: Instant molecular orientation microscopy for the life sciences. bioRxiv 2023.02.07.527479 (Feb 2023), doi: https://doi.org/10.1101/2023.02.07.527479

[STEPS]
SECTION: Protocol
1. Argon plasma clean cover glass (VWR collection, 631-0124) for 30 min in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).

SECTION: Protocol
3. Create a sample well on the cleaned cover glass by sticking a frame-seal slide chamber (9x9 mm, SLF0201, Bio-rad) on the cover glass.
SECTION: Protocol
5. Use a pipet to remove the excess PLL from the well and immediately replace it with 70 µL of filtered PBS.
 


SECTION: Protocol
4. Pipet 70 µL of 0.01% PLL (0.01% poly-L-lysine solution, P4707, Sigma-Aldrich) into the well and wait for 15 min. The PLL will coat the surface of the cover glass.

 

SECTION: Protocol
6. Use a pipet to remove the excess filtered PBS from the well and immediately replace with70 µL filtered PBS. Gently pipet up and down in the corners of the well. Repeat this step 2 more times.

SECTION: Protocol
7. Use a pipet to remove the excess PBS from the well and immediately replace with 50 µL of 1 nanomolar (nM) SYTOX Orange (S11368, Invitrogen). The SYTOX Orange molecules will stick to the surface of the PLL-coated cover glass.

SECTION: Protocol
8. Image the sample straight away and make sure it doesn't dry out during imaging.
SECTION: Protocol
2. In the meantime:
Filter phosphate-buffered saline (PBS) using a 0.02 μm syringe filter (6809-1102, Whatman).
Dilute SYTOX Orange (S11368, Invitrogen) in filtered PBS to a concentration of 1 nanomolar (nM).
TITLE: Test protocol II
AUTHORS: Abby   Moore

[DESCRIPTION]
This is a test protocol

Here's an protocol reference:


 


Here's a citation:


 


[BEFORE_START]
This is what you should know before you start


 


[GUIDELINES]
Responsibilities....

[STEPS]
1. Use 80:20 MeOH:H2O for this step. This is not easy to access by machine.
2. If you haven't already, make a solution with the following components:

80 % volume   
20 % volume   

3. If you haven't already, make a solution with the following components:

80 % volume   
20 % volume   

4. Use this piece of equipment:

 


5. 
 






6. My image saved to GDrive.
TITLE: Protocol of data recording for the recognition of familiar speech in the presence of competitive noise
AUTHORS: Kelly Andrade, Thamyres Ataíde Bezerra Verçosa, Aline Tenório Lins Carnaúba, Pedro Menezes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for data collection of speech recognition in noise. The protocol has, general data fields, information about the speaker, audiometric data and speech recognition data with different intensities.</div></div>

[STEPS]
TITLE: Fecobionics Test in Normal Subjects 
AUTHORS: Yanmin Wang, Hans Gregersen

[DESCRIPTION]
It is the protocol of Fecobionics test in normal subjects. Briefly, the device will be inserted into participants' rectum and several motions (cough, squeeze, straining, push) will be done. After the motions, the bag will be inflated and the participant may have different sensations during inflation. Data will be recorded and shown in real time.

[STEPS]
1. Make sure the participant is eligible to do the study, and make sure he/she understands the study and has signed the informed consent form.
2. Ask the subject to empty bladder and rectum before the test.
3. Participant changes clothes and lies on left side.
4. Calibrate the Fecobionics device and make sure the transmission signal is good.
5. Fully lubricate Fecobionics device. Insert device gently into rectum, following a finger examination.
6. Participant moves to commode, in sitting position. Wait up to 5 mins until pressure data is stable.
7. Ask the participant to cough twice, with 15-30 seconds in between.
8. Ask the participant to squeeze three times, 5 seconds for each time, and 15-30 seconds in between. 
9. Ask the participant to do a long squeeze that lasts 30 seconds.
10. Ask the participant to do a straining (contract both abdominal and anal muscles) that lasts 20 seconds.
11. Ask the participant to push three times, 15 seconds for each time, and 30 seconds in between. 
12. Inflate the balloon with warm saline. Ask the participant to report sensations.
13. Stop inflating when the participant feels urge, or the maximum volume (100ml) is reached.
14. Detach the filling tube and give the participant privacy to defecate. If the participant cannot defecate Fecobionics in 3 minutes, withdraw it manually by pulling the thread attached to the device.
15. Test complete. Save data and clean up.
TITLE: Primer design using NIH&#39;s Primer-BLAST tool.
AUTHORS: Pedro C. Díaz, Francisco Franco García Calderon

[DESCRIPTION]
This protocol demonstrates the basic usage of the Primer-BLAST tool in order to design PCR primers from a given FASTA sequence.
The results should show the sequence of at least one pair of primers (forward and reverse) within the chosen parameters.

[STEPS]
SECTION: Go to NIH&#x27;s Primer-BLAST tool
1. Open your internet browser (Google Chrome recommended).

SECTION: Go to NIH&#x27;s Primer-BLAST tool
2. Go to https://www.ncbi.nlm.nih.gov/tools/primer-blast/
SECTION: Enter FASTA sequence
3. 
 
Copy and paste your FASTA sequence in the text box or upload a FASTA file using the ¨Choose File¨ button.
SECTION: Enter primer parameters
5. 
 
Enter the optimal parameters for your primers. Depending on your application, the PCR product size and the primer Melting temperatures are extremely important values!
SECTION: Enter FASTA sequence
4. You can select a custom range (i.e. the positional value of the first and last nucleotide of your ROI) for the forward and reverse primers.
SECTION: Analyze
6. 
 
Click on the ¨Get Primers¨ button.