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

[GUIDELINES]
<p>A genetic transformation method has been established in <em>Perkinsus marinus</em> using the Amaxa Nucleofector system (Fernández-Robledo et al. 2008, Sakamoto et al. 2016). We tried to optimize the transformation conditions for <em>P. marinus</em> using the Gene Pulser Xcell (Bio-Rad) system. We determined an optimal condition using FITC-dextran introduction tests: 200 V, 15 ms square wave in Bio-Rad 0.2 cm cuvette with 100 µL modified cytomix solution (70 mM KCl, 5.8 mM potassium phosphate buffer, 2.9 mM MgCl2, 14.5 mM HEPES, 0.09 mM CaCl<sub>2</sub>, 1.2 mM EGTA, 0.2 M mannitol, pH 7.4).</p>
<p> </p>
<p>We compared the efficiency of FITC-dextran introduction between two different systems of Amaxa (parasite nucleofector solution 2 with the program D-023) and Bio-Rad (described above). We detected that more than 50% of cells showed positive fluorescence in the both cases (Fig. 1 and 2).</p>
<p> </p>
<p>Fig. 1: FITC fluorescence in the Amaxa condition</p>
<p><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dyzdt4e.jpg" width="200" height="200" data-src="https://s3.amazonaws.com/pr-journal/dyydt4e.jpg" data-ofn="Ama1.jpg" /></p>
<p> </p>
<p>Fig. 2: FITC fluoresncence in the Bio-Rad condition</p>
<p><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dy3dt4e.jpg" width="200" height="200" data-src="https://s3.amazonaws.com/pr-journal/dy2dt4e.jpg" data-ofn="Bio1.jpg" /></p>
<p> </p>
<p> </p>
<p><em>P. marinus </em>cells were transiently transformed with pMOE-GFP by the both conditions, and we observed GFP fluorescence 48 h after transformation. Transformation efficiency of the Amaxa condition was more than 100 times higher than the Bio-Rad condition, although the FITC-dextran tests showed the similar efficiency (Fig. 3 and 4). I guess FITC-dextran introduction efficiency would not be correlated with actual transformation efficiency.</p>
<p> </p>
<p> </p>
<p>Fig. 3: GFP expressing cells transformed by the Amaxa condition</p>
<p><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dyvdt4e.jpg" data-src="https://s3.amazonaws.com/pr-journal/dyudt4e.jpg" data-ofn="Amaxa_01.jpg" /></p>
<p> </p>
<p>Fig. 4: GFP expressing cells transformed by the Bio-Rad condition</p>
<p><img class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dyxdt4e.jpg" data-src="https://s3.amazonaws.com/pr-journal/dywdt4e.jpg" data-ofn="BioRad_02.jpg" /></p>

[STEPS]
TITLE: A lexical learning task for English and Italian participants
AUTHORS: Chiara Valeria Marinelli, Pierluigi Zoccolotti, Cristina Romani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>“Lexical learning” is the ability to set up stable and accurate mental representations of words in the lexicon  (Di Betta & Romani, 2006). The task allow to test the ability of Italian and English subjects to learn </span><span style = "font-style:italic;">written</span><span> pseudo-words in an explicit paired associate learning task, through corrective feedback. For more details see Marinelli, Zoccolotti & Romani (in revision). </span></div></div>

[STEPS]
TITLE: RNA Extraction and RT-qPCR
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to extract RNA from mouse brain tissue and quantify with RT-qPCR.

[STEPS]
1. Homogenize hemibrain in 3mL 1X PEPI buffer using a dounce homogenizer.
1X PEPI Buffer: 5mM EDTA + 1X protease inhibitor in 1X phosphate buffered saline (PBS)
2. Take 3% of homogenate and add 1mL TRIzol Reagent and follow their user guide.
3. Synthesize cDNA with 5X All-In-One RT Master Mix Kit then perform qPCR.
qPCR settings: 95oC for 5 minutes, 40x (95oC for 15 seconds then 60oC for 60 seconds), melting curve
TITLE: Supplemental Resources for You Can’t Hide Your Lying Eyes: Honesty Oaths and Misrepresentation
AUTHORS: J Jobu Babin, Haritima Chauhan, Feng Liu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Abstract</div><div class = "text-block">Lying about personal qualifications for a job or in college admissions is common and has recently become a high profile issue. In this paper, we explore the decision to misrepresent one’s self and subsequently determine whether honesty oaths impact truthful reporting of a personal characteristic. To do this, we execute a field experiment on Amazon MTurk using a self-reporting task involving worker eye color. We provide empirical evidence that honesty oaths elicit more truthful behavior, primarily impacting “extreme” lies. Oath-taking decreased the cost associated with misreporting personal characteristics by 70%.  Oaths marginally decrease shirking.</div></div>

[STEPS]
TITLE: Primer stock preparation
AUTHORS: Brian Lovett, Kristen Pierce

[DESCRIPTION]
General protocol for preparation of lyophilized primer stocks from IDT.

[GUIDELINES]
Recommended: When you receive your lyophilized primers, check the name of the primer, primer sequence and the concentration listed on the tube. IDT provides a specification sheet you can download/archive for more information.

[STEPS]
SECTION: Preparation of stock solution
1. Reconstitute lyophilized primers in required volume of molecular grade water (i.e., RNase/DNase free) to 100 micromolar (µM).

SECTION: Preparation of working solution
4. Allow stock solution to thaw completely at Room temperature.

SECTION: Use of primers
8. For a typical 25 µL PCR reaction, 1 µL of each primer is used (i.e., 0.4 micromolar (µM) final concentration).
SECTION: Preparation of stock solution
2. After water has been added, vortex for at least 10 s to ensure the lyophilized primers are fully dissolved. 
SECTION: Preparation of stock solution
1.1. Practically, the microliters of water required to create a 100 µM solution is 10x the nmol concentration of lyophilized primer (i.e., if 23.5 Nanomoles is noted on side of tube, then add 235 µL of water to stock tube).

SECTION: Preparation of stock solution
3. Once reconstituted, the stock solution can be stored in a labeled box at -20 °C, and thawed completely before use. 

SECTION: Preparation of working solution
5. Vortex the stock solution for at least 10 s to ensure homogenization before adding it to the working solution. 
SECTION: Preparation of working solution
7. Once the concentrated stock is added, vortex the new working solution for at least 10 s and store at -20 °C to use as needed.  

SECTION: Use of primers
8.1. To maintain optimal use of your primers, limit freeze/thaw cycles, as this can lead to degradation over time. 
SECTION: Preparation of working solution
4.1. While waiting for your stock to thaw, prepare clean microcentrifuge tubes by labeling them with the primer name and date of preparation. Label the lid so it is easy to identify tubes in freezer box.
SECTION: Preparation of working solution
6. To prepare a 10 Molarity (m) working solution from the 100 micromolar (µM), you will dilute 1:10  (i.e., 1 µL of stock for every 9 µL of water). 

SECTION: Preparation of working solution
6.1. For a 100 µL working solution, pipette 90 µL of molecular grade water into the newly labeled microcentrifuge tube and then add 10 µL of the stock solution.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is the general immunofluorescence protocol used in the Laboratory for Integrative Neuroscience at NIAAA. This protocol can be adapted for most primary antibodies but specific antibodies and concentrations need to be determined by the user and the protocol adapted accordingly. Secondary antibodies listed are optimal for the Axiozoom and LSM880 filters and excitation sources and may need to be changed to match the microscope available. I also provide tips for optimizing staining, reducing background, and characterizing antibodies.</p>

[BEFORE_START]
<ul>
<li><span style="display: inline !important; float: none; background-color: transparent; color: #000000; font-family: 'Open Sans',sans-serif; font-size: 13px; font-style: normal; font-variant: normal; font-weight: 400; letter-spacing: normal; line-height: 16px; orphans: 2; text-align: left; text-decoration: none; text-indent: 0px; text-transform: none; -webkit-text-stroke-width: 0px; white-space: normal; word-spacing: 0px;">If unfamiliar with fluorescence microscopy, see tutorial:  https://micro.magnet.fsu.edu/primer/techniques/fluorescence/fluorhome.html</span></li>
<li>Before starting, validate and titrate all antibodies. Select concentrations that result in a strong signal that does not saturate and does not result in non-specific staining. The titration should result in a signal that is dependent on the concentration of the primary antibody such that a change in concentration result in a change in exposure time when imaging (i.e. 1:1000 is a 1 second exposure, while 1:2000 requires 2 seconds of exposure. Details on titration are provided in the protocol.  </li>
<li><span style="display: inline !important; float: none; background-color: transparent; color: #000000;">If possible, test in a knock out.</span></li>
<li>Our preferred sources for primary antibodies include Millipore, Frontier Biosciences (Japan), Immunostar, Abcam, Encor, Alamone, and Cayman.  </li>
<li> Order the cross-adsorbed, purified versions of the secondary antibodies and carefully titrate using tissue with no primary antibody.  We generally use them between 1:1000 and 1:3000. Test secondaries in combination for cross reactivity in multi-labels. Mouse and rat will cross react.</li>
</ul>

[STEPS]
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TITLE: Passaging and plating A549 cells
AUTHORS: ATCC, Steven Walter

[DESCRIPTION]
Volumes used in this protocol are for 75 cm2flask; proportionally reduce or increase amount of dissociation medium for culture vessels of other sizes. Corning T-75 flasks (catalog #430641) are recommended for subculturing this product.

[BEFORE_START]
Warm all solutions to 37 deg C and prepare biosafety cabinet by starting the blower and wiping down all surfaces with 70% ethanol.  Organize your workspace in the biosafety cabinet to have all clean/fresh reagents on one side and all waste on the other.  Anything you will be taking into the biosafety cabinet should also be wiped clean using 70% ethanol and paper towels

[GUIDELINES]
Most human cell lines are vulnerable to infection by the microbes on your skin, be careful to work aseptically to avoid contaminating your cultures and keep yourself safe. 

[STEPS]
SECTION: Splitting A549 in T-75
2. Remove and discard culture medium, by tipping dish carefully to one side and aspirating the media without disturbing the adherent cells
SECTION: Splitting A549 in T-75
3. Briefly rinse the cell layer with 1.0 mL 0.05% (w/v) Trypsin - 53 mM EDTA solution to remove all traces of serum which contains trypsin inhibitor.

SECTION: Splitting A549 in T-75
4. Add 2.0 mL of Trypsin-EDTA solution to flask and observe cells under an inverted microscope until cell layer is dispersed (about 10 min). Note: To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. Cells that are difficult to detach may be placed at 37°C to facilitate dispersal. 

SECTION: Splitting A549 in T-75
5. Add 6 mL of complete growth medium and aspirate cells by gently pipetting. 

SECTION: Splitting A549 in T-75
1. Warm all solutions to 37 °C 
SECTION: Splitting A549 in T-75
6. Remove 10 µL of cell suspension for the hemocytometer. Average the count of the four-corners and multiply by 10,000 (104) to get concentration (cells/mL).

SECTION: Splitting A549 in T-75
8. Seed new plates with the recommended amount from the table.
 


SECTION: Splitting A549 in T-75
7. Centrifuge 150 x g, 5 min ; Remove supernatant. Resuspend in 6 mL fresh media.
SECTION: Splitting A549 in T-75
9. Incubate at 37 °C for approximately 2880 min
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>mothur is a bioinformatics tool for analyzing 16S rRNA gene sequences, which can be used to process data generated by Sanger, PacBio, IonTorrent, 454, and Illumina (MiSeq/HiSeq). </p>

[BEFORE_START]
<p>Before starting, please visit the ECOGEO website for more information on this "Introduction to Environmental 'Omics" training series. The site contains a pre-packaged virtual machine that can be downloaded and used to run all of the protocols in this protocols.io collection. In addition to the VM, the website contains video and presentations from our initial "Intro to Env 'Omics" workshop held at the Univ. of Hawai'i at Manoa on 25-26 Jul 2016.<br /><br />Please email ‘ecogeo-join@earthcube.org’ to join the ECOGEO listserv for future updates.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Angionesis </span><em><span style="font-weight: 400;">in vitro: </span></em><span style="font-weight: 400;">Es la formación </span><em><span style="font-weight: 400;">In vitro </span></em><span style="font-weight: 400;">de tubos capilares por células endoteliales en la matriz de una membrana basal, es un metodo </span><em><span style="font-weight: 400;">in vitro </span></em><span style="font-weight: 400;">poderoso para evaluar varios factores que promueven o inhiben angiogenesis. Es para definir las rutas de señalización en angiogenesis, identificando los genes reguladores de angiogenesis y caracterizando las células endoteliales del progenitor.</span></p>
<p><span style="font-weight: 400;">El ensayo puede ser hecho como primera valoración antes de una prueba costosa con animales y puede ser hecho como un proceso de alta tecnología. Este es un método </span><em><span style="font-weight: 400;">in vitro </span></em><span style="font-weight: 400;">privilegiado para evaluar reguladores angiogenicos.</span></p>
<p> </p>
<p><span style="font-weight: 400;">La realizacion de este protocolo fue posible gracias al apoyo del departamento administrativo de ciencia tecnología e innovacion, Colciencias a traves del proyecto 111356933173 convocatoria569-2012. </span></p>
<p> </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[STEPS]
SECTION: Recovering skeletons
?. 
SECTION: Rinsing
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SECTION: Cleaning
?. 
SECTION: Cleaning
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SECTION: Diluting
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SECTION: Rinsing
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SECTION: Staining
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SECTION: Staining
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SECTION: Diluting
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SECTION: Rinsing
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SECTION: Preparing for imaging
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SECTION: Preparing for imaging
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SECTION: Imaging
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TITLE: Cell culture
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes routine cell culture maintenance.

[STEPS]
SECTION: Cell culture
1. Grow HeLa and HEK293T cells in Dulbecco Modified Eagle Medium (DMEM, Thermo
Fisher) supplemented with 10% (v/v) Fetal Bovine Serum (FBS, Thermo Fisher), 25 millimolar (mM) HEPES (Thermo Fisher), 1% (v/v) non-essenMal amino acids (NEAA, Thermo Fisher), and 1% (v/v) Penicillin-Streptomycin (Thermo Fisher).
SECTION: Cell culture
2. Culture HAP1 cells in Iscove’s Modified Dulbecco’s Medium (IMDM, Thermo Fisher), supplemented with 10% (v/v) Fetal Bovine Serum (FBS, Thermo Fisher) and 1% (v/v) Penicillin-Streptomycin (Thermo Fisher).
SECTION: Cell culture
3. Regularly test all cell lines for mycoplasma contaminations.
SECTION: Cell culture
4. Split the cells by trypsinization once confluent.
SECTION: Cell culture
5. To this end, remove medium with Pasteur pipette. 
SECTION: Cell culture
6. Wash cells with 1x PBS and then add trypsin for 5 min at 37 °C.
SECTION: Cell culture
7.  Afterwards, resuspend cells in medium and retain 10% for further culturing and expansion. 
SECTION: Cell culture
8. Discard the rest or collect for seeding for experiments.
TITLE: Primary neuronal cultures
AUTHORS: Miguel Da Silva Padilha, Irina Dudanova, F. Ulrich Hartl, Itika Saha, Mark S. Hipp

[DESCRIPTION]
This protocol describes the preparation of primary neuronal cultures from E15.5 CD-1 wild type mouse embryos. Experiments involving animal models must be performed in accordance with relevant institutional guidelines and regulations.

[STEPS]
1. Sacrifice pregnant female mice by cervical dislocation.
2. Remove the uterus from the abdominal cavity and place into a 10 cm sterile Petri dish on ice containing dissection medium, consisting of Hanks’ balanced salt solution (HBSS) supplemented with 0.01 M HEPES, 0.01 M MgSO4 and 1% penicillin/streptomycin.
3. Isolate each embryo, decapitate the heads, remove the brains from the skull and immerse in ice-cold dissection medium.
4. Dissect cortical hemispheres, and remove meninges under a dissection microscope.
5. Collect the cortices in a 15 mL sterile tube and digest with 0.25% trypsin containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 15 μL 0.1% DNAse I for 20 min at 37 °C.
6. Stop digestion by removing the supernatant and washing the tissue twice with Neurobasal medium (Invitrogen) containing 5% Fetal Bovine Serum.
7. Resuspend the tissue in 2 mL Neurobasal medium and triturate to achieve a single cell suspension.
8. Spin cells at 130 x g, remove the supernatant, and resuspend the cell pellet in Neurobasal medium with 2% B-27 supplement (Invitrogen), 1% L-glutamine (Invitrogen) and 1% penicillin/streptomycin (Invitrogen).
9. Plate cells at desired density in dishes or coverslips coated with 1 mg/mL poly-D-lysine (Sigma) and 1 µg/mL laminin (Thermo Fisher Scientific).
TITLE: coloMOCA implantation protocol  
AUTHORS: Brett Hanzlicek, Dennis Bourbeau

[DESCRIPTION]
This protocol describes the implantation of our bowel device (ColoMOCA) in pigs.

[STEPS]
SECTION: Anesthesia
1. Anesthetize/prep the animal for surgery.

       Aseptic procedures by trained personnel are utilized during the surgery. All tools are sterilized         prior to insertion. Drapes will be used and personnel performing surgery will wear clean surgical scrubs, and sterile gowns/gloves and masks.
SECTION: Laparotomy
2. Make a midline suprapubic incision ~9 cm long to expose the peritoneal cavity and viscera.
SECTION: Colotomy
3. Make a single, small incision into the colon.
SECTION: Insert and anchor ColoMOCA
4. Insert device through the small incision and secure in place with sterile non-absorbable sutures.
SECTION: Test Wireless Transmission
5. Wake the coloMOCA with radio if not already awake.  Confirm data transmission.
SECTION: Close incision
6. Close the colon with an absorbable suture in an interrupted pattern
Close the peritoneum and fascia with absorbable sutures using a continuous suture pattern.
Close the subcutaneous tissue and skin with non-absorbable monofilament suture with a cutting needle, using an interrupted suture pattern.Skin staples may also be used.
SECTION: Test Wireless Transmission (2)
7. Confirm data transmission and then put device to sleep
SECTION: CT-Imaging
8. Image to verify placement of the sensor.
SECTION: Place jacket on the pig
9. Place and zip the jacket on the pig while still anesthetized.
SECTION: Wake up the animal
10. Stay with the animal until fully awake.  Return to pig room.
TITLE: qPCR quantification of methanogens using general mcrA primers
AUTHORS: Roey Angel, Eva Petrova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>General </span><span style = "font-style:italic;">mcrA</span><span> primers for quantifying methanogens using a SYBR Green-based assay.</span></div><table border><tr style = "text-align:center;"><td>  </td><td>A</td><td>B</td><td>C</td><td>D</td></tr><tr><td style = "text-align:center;">1</td><td rowspan = "" colspan = "" style ="display : table-cell;">Name</td><td rowspan = "" colspan = "" style ="display : table-cell;">Direction</td><td rowspan = "" colspan = "" style ="display : table-cell;">Sequence</td><td rowspan = "" colspan = "" style ="display : table-cell;">Citation</td></tr><tr><td style = "text-align:center;">2</td><td rowspan = "" colspan = "" style ="display : table-cell;">mlas_mod</td><td rowspan = "" colspan = "" style ="display : table-cell;">Fwd</td><td rowspan = "" colspan = "" style ="display : table-cell;">GGY GGT GTM GGD TTC ACM CAR TA</td><td rowspan = "" colspan = "" style ="display : table-cell;">Angel et al., 2011</td></tr><tr><td style = "text-align:center;">3</td><td rowspan = "" colspan = "" style ="display : table-cell;">mcrA_rev</td><td rowspan = "" colspan = "" style ="display : table-cell;">Rev</td><td rowspan = "" colspan = "" style ="display : table-cell;">CGT TCA TBG CGT AGT TVG GRT AGT</td><td rowspan = "" colspan = "" style ="display : table-cell;">Steinberg and Regan, 2008</td></tr></table></div>

[STEPS]
?. [QPCR mixture]
ABCD1ReagentFinal conc.1 tube (25 μl)plate (25 μl x 100)2PCR H2O&nbsp;2.852853qPCR master mix1x12.512504MgCl2 (25mM)*3.5 mM3.53505BSA (50 μg μl-1)0.8 μg μl-10.4406mlas-mod (25 μM)0.25 μM0.25257mrcA-rev (25 μM)0.25 μM0.25258Template&nbsp;55 x 100 * Different master mixes vary in concentration of MgCl2 and the amount should be adjusted accordingly
ABCD1ReagentFinal conc.1 tube (25 μl)plate (25 μl x 100)2PCR H2O&nbsp;2.852853qPCR master mix1x12.512504MgCl2 (25mM)*3.5 mM3.53505BSA (50 μg μl-1)0.8 μg μl-10.4406mlas-mod (25 μM)0.25 μM0.25257mrcA-rev (25 μM)0.25 μM0.25258Template&nbsp;55 x 100
?. [Programme]
1.  94◦C – 5′2.  x 40 {       a.  95◦C – 30′′       b.  57◦C – 45′′       c.   72◦C – 30′′       d.  84°C -  10'' take a snapshot      }3.  Melt curve:  75°C – 94.8°C –6’’. Increase by 0.2°C
TITLE: Protocol-IHC
AUTHORS: Klaus Hirschbühl

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center">Protocol-IHC to the paper:</div></div><div class = "text-block"><div class = "justify" style = "text-align:center"></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Viral mapping in COVID-19 deceased in the Augsburg autopsy series of the first wave: a multiorgan and multimethodological approach</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;">Full organ mapping of patients with COVID-19: an autopsy study</span></div></div></div>

[STEPS]