eamag/protocols-with-steps huggingface dataset:
TITLE: Rearing Ambrosia Beetles in Media
AUTHORS: James Skelton, Sawyer Adams, Andrew J. Johnson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to produce a supply of living beetles in various developmental stages in the laboratory. </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: https://www.jpost.com/promocontent/keto-extreme-fat-burner-south-africa-price-at-clicks-dischem-denmark-living-lifestyle-705766
AUTHORS: ketoextremefatburnerCOST  

[DESCRIPTION]
Keto Extreme Fat Burner requires some genuine festival now and can be bought without a doubt in the least demanding ever way.

[STEPS]
TITLE: Sensor Guided Needle Entry Procedures: A Scoping Review of Advancements to the Veress Needle &amp; Selected Needle Entry Techniques
AUTHORS: Chimwemwe Miti, Richard Scott, Hermes Gadelha-Bloomfield

[DESCRIPTION]
Primary entry during minimally invasive abdominal surgery remains the one step associated with controversy and complications. 
This scoping review attempts to draw literature describing sensor augmented techniques for introducing the Veress needle into the abdomen in a bid to reduce associated challenges. Publications related to other relevant needle entry techniques into critical spaces where sensors are successfully utilised are also briefly reviewed. 
It is expected that an overview of related areas in which the technology is applicable and modifications to established techniques alongside the added improvements will be described.

[BEFORE_START]
None

[GUIDELINES]
Not applicable

[STEPS]
SECTION: Eligibility Criteria
1. Studies in English
SECTION: Eligibility Criteria
2. Publication period 1992- May 2024
SECTION: Eligibility Criteria
3. Articles with full text available
SECTION: Eligibility Criteria
4. Human subjects
TITLE: The calculation of gut metabolic modules from gene profile
AUTHORS: Qi Wang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The calculation of gut metabolic modules from gene profile</div></div>

[STEPS]
?. Step 1: the putative amino acid sequences were translated from the gene catalogues and aligned against the proteins/domains in the KEGG databases (release 79.0, with animal and plant genes removed) using BLASTP (v2.2.26, default parameter except that -e 0.01 -b 100 -K 1 -F T -m 8). Each protein was assigned to the KO group by the highest scoring annotated hit(s) containing at least one HSP scoring >60 bits.The relative abundance profile of KOs was determined by summing the relative abundance of genes from each KO.
?. Step 2: The calculation of gut metabolic modules by one command:'java -jar gmms.jar -a 2 -d GMMs.v1.07.txt -i input_KO_profile -s average -o index'You could refer to the http://www.raeslab.org/companion/gmms/ for 'gmms.jar' and 'GMMs.v1.07.txt'.
TITLE: QIAGEN DNeasy PowerSoil Kit
AUTHORS: QIAGEN

[DESCRIPTION]
For the isolation of microbial genomic DNA from all soil types

The DNeasy PowerSoil Kit comprises a novel and proprietary method for isolating genomic DNA from environmental samples using patented Inhibitor Removal Technology® (IRT). This kit is intended for use with environmental samples containing high humic acid content, including difficult soil types such as compost, sediment and manure. Other more common soil types have also been used successfully with this kit. The isolated DNA has a high level of purity, which allows for more successful PCR amplification of organisms from the sample. PCR analysis has been performed to detect a variety of organisms including bacteria (e.g., Bacillus subtilis, Bacillus anthracis), fungi (e.g., yeasts, molds), algae and actinomycetes (e.g., Streptomyces).

Protocol used successfully to detect target fish species sedDNA by Olajos et al., 2018; Nelson-Chorney et al., 2019; Thomson-Laing et al., 2020; Shiragaki et al., 2021; and Naro-Maciel et al., 2022 

[BEFORE_START]
Perform all centrifugation steps at room temperature (15–25°C).
If Solution C1 has precipitated, heat at 60°C until precipitate dissolves.

[STEPS]
SECTION: Sample preparation &amp; cell lysis
1. ADD 0.25 g of soil sample to the PowerBead Tube 

VORTEX gently to mix
SECTION: Sample preparation &amp; cell lysis
2. ADD 60 µL of Solution C1 

INVERT several times or vortex briefly
SECTION: Sample preparation &amp; cell lysis
3. SECURE PowerBead Tubes horizontally using a Vortex Adapter for 24 (1.5–2.0 ml) tubes 

VORTEX at maximum speed for 10 min 
SECTION: Sample preparation &amp; cell lysis
4. CENTRIFUGE tubes at 10.000 x g for 30 s 

TRANSFER supernatant to a clean 2 mL Collection Tube
SECTION: Inhibitor removal
5. ADD 250 µL of Solution C2 

VORTEX for 5 s 

INCUBATE at 2 °C to 8 °C for 5 min
SECTION: Inhibitor removal
6. CENTRIFUGE tubes at 10000 x g for 1 min 

AVOIDING the pellet, transfer up to 600 µL of supernatant to a clean 2 mL Collection Tube 
SECTION: Bind DNA
9. SHAKE to mix Solution C4

ADD 1200 µL of Solution C4 to the supernatant

VORTEX for 5 s

SECTION: Bind DNA
10. LOAD 675 µL onto an MB Spin Column

CENTRIFUGE at 10.000 x g for 1 min 

DISCARD  liquid flow-through
SECTION: Bind DNA
11. REPEAT step 10 twice, until all of the sample has been processed
SECTION: Wash spin column
12. ADD 500 µL of Solution C5

CENTRIFUGE at 10.000 x g for 30 s 

DISCARD liquid flow-through
SECTION: Wash spin column
13. CENTRIFUGE again at 10.000 x g 1 min 

CAREFULLY place the MB Spin Column into a clean 2 mL Collection Tube. Avoid splashing any residual Solution C5 onto the column
SECTION: Elute the DNA
14. ADD 100 µL of Solution C6 to the center of the white filter membrane

CENTRIFUGE at 10000 x g for 30 s 

DISCARD the MB Spin Column

DNA is now ready for downstream applications
SECTION: Inhibitor removal
7. ADD 200 µL of Solution C3

VORTEX briefly to mix

INCUBATE at 2 °C to 8 °C for 5 min 
SECTION: Inhibitor removal
8. CENTRIFUGE tubes at 10000 x g for 1 min 

AVOIDING the pellet, transfer up to 750 µL of supernatant to a clean 2 mL Collection Tube
TITLE: Vivarium Population Spenser: Emigration protocol
AUTHORS: Camila Rangel Smith, Kasra Hosseini

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Description of the steps followed by Vivarium Population Spenser library when running the Emigration module. </div></div>

[STEPS]
?. For each time step:
?. Divide the annual international emigration rates for that local authority by the number of time steps existing in a year.
?. Select all individuals in the sample that appear as "alive" and have an associated gender.
?. For these individuals, get the emigration rate given their age, gender, ethnicity and location and turn it into a probability.
?. Using random sampling and the emigration probability of the individuals, choose which individuals emigrate in that time step.
?. Change the status of these individuals from "alive" to "emigrated". Record the time when they emigrated.
TITLE: Basic Cell Culture Maintenance: Splitting Cells
AUTHORS: leroi.bwh

[DESCRIPTION]
Basic protocol to split THP1

[BEFORE_START]
Make complete DMEM:
 
   Reagent Volume   DMEM 432.5 mL   FBS 50 mL   Pen/Strep 5 mL   HEPES (1M, pH 7.4) 12.5 mL   
 

[STEPS]
SECTION: Preparation
1. Confirm that cells are at least 80% confluent by microscopy.
 
SECTION: Remove Media
5. Aspirate the media from the flask using a sterile autoclaved glass pipette. Do not touch the cells with the pipette. 
 
SECTION: Transfer
6. Transfer ALL contents/cells to a 50 mL falcon tube.

SECTION: Spin
7. Spin down 130 rcf for 7 minutes.7 min130 x g, 37 °C 

SECTION: Spin
8. While spinning, clean surfaces with EtOH and label new flasks, noting the +1 passage number and dilution.
SECTION: Remove Media
9. Aspirate media from falcon tubes with cells; make sure to not disturb the pellet.
SECTION: Resuspend Pellet
10. Add 5 mL media to pellet and pipette violently up and down.

SECTION: Prepare New Flask
14. Add the required amount of medium.

SECTION: Incubate
16. Gently shuffle, ensure even dispersal, and return the fresh flask to incubator.
SECTION: Preparation
3. UV light for 30 minutes then spray down the biosafety cabinet with 70% ethanol and use  as a secondary decontaminant.
SECTION: Preparation
2. Warm complete RPMI in 37°C water bath.
SECTION: Count the cells
11. Pipette 20 µL and add them to the Trypan blue


SECTION: Preparation
4. Prepare 20 µL of Trypan blue into a 200 µl Eppendorf.

SECTION: Count the cells
12. Count the cells using a Neubauer counting chamber

    

SECTION: Count the cells
13. Calculate the required amount of medium to keep a concentration of 100 kC/ml 

SECTION: Prepare New Flask
15. Distribute in flasks, no more than 50 mL per flask.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>we generated five clonal, axenic strains of Symbiodinium. These strains were assigned to clades A (two strains), B, E, and F based on their chloroplast 23S rDNA</p>
<p>sequences. Growth studies in liquid cultures showed that the clade B strain and one of the clade A strains were able to grow photoautotrophically (in light with</p>
<p>no fixed carbon), mixotrophically (in light with fixed carbon), or heterotrophically (in dark with fixed carbon). Here this protocol describes the steps of culturing SSB01 in different types of medium as an example.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Stereotaxic Injection by Iontophoresis
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the delivery of a neuronal tracer using the iontophoretic method. The surgery uses a stereotaxic system to target specific brain coordinates in the mouse.  

Note: 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.

[STEPS]
TITLE: TRANSDUCTION OF i3NEURONS (Support Protocol 4)
AUTHORS: Michael S. Fernandopulle, Ryan Prestil, Christopher Grunseich, Chao Wang, Li Gan, Michael E. Ward

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lentiviral infection of neurons can be used for a variety of applications in microscopy and biochemistry, provided that the transgene is driven by promoters not silenced in iPSCs or neurons (e.g., CAG, PGK In EF-1α, not CMV, see Critical Parameters and Troubleshooting). The precise parameters for optimal viral production and supernatant collection/concentration will depend on the type of virus used, viral packaging mechanism, viral packaging cell line, and specific transgene introduced. Highly efficient results have been observed with lentivirus produced by Lenti-X HEK cells, with transgenes carried in the pLEX backbone.</div></div>

[STEPS]
?. [Day 0]
Coat a 6-well dish with PLO (see Basic Protocol 6).
?. [Day 1]
Check cells for confluency under an inverted microscope. Cells should be > 95 % confluent for maximal viral production efficiency
If necessary, wait a day to transfect so that the cells are nearly confluent.
?. [Day 0]
Plate 2.5 × 106 Lenti-X HEK cells in 6-well dish well in  DMEM/F12 containing 10 % FBS (HEK medium).
1.5 ml
?. [Day 1]
Warm two tubes with  Opti-MEM each to .
150 µl
0 Room temperature
?. [Day 1]
Add  psPAX,  pMD302,  pAdvantage,  pLEX viral vector,and  P3000 reagent to tube A.
1.6 µg
0.6 µg
0.2 µg
2.4 µg
10 µl
?. [Day 1]
Add  Lipofectamine 3000 to tube B.
1.87 µl
?. [Day 1]
Incubate tubes at  for .
0 Room temperature
?. [Day 1]
Add the contents of tube A into tube B with a P200.
?. [Day 1]
Incubate tube at  for  to .
0 Room temperature
?. [Day 1]
Add contents of tube dropwise to to the medium of Lenti-X cells with a P1000.
?. [Day 1]
Agitate plate to evenly distribute transfection solution.
?. [Day 2]
Replace medium on Lenti-X HEK cells with  of warm, fresh HEK medium supplemented with of 500× ViralBoost reagent.
3 ml
6 µl
?. [Day 2]
Check cells with a fluorescent microscope to ensure expression of any fluorescent proteins in the viral vector.
?. [Day 4]
Check cells under inverted microscope for multinucleated morphology (indicating viral production) and fluorescent protein production.
?. [Day 4]
Collect medium from well.
?. [Day 4]
Optional: Replace medium on HEK cells with  to  fresh HEK medium, to be collected the following day. This process can be repeated until Day 6 to collect more virus. Be aware that viral titer decreases with each collection. Lentiviral preps can also be concentrated using various commercially available reagents (e.g., Clontech, cat. no. 631231).
1 ml
2 ml
?. [Day 1]
Gently flick to mix.
?. [Day 1]
Flick to mix.
?. [Day 1]
Immediately flick the tube several times to mix.
?. [Day 4]
Filter to purify virus from floating HEK cells.
Instead of filtering, medium may be centrifuged at >  for    at   to remove cells/debris, followed by transferring the supernatant to a new microcentrifuge tube.
?. [Day 4]
Either use immediately or freeze aliquots at .
-80 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is our transfer protocol for the transfer of proteins using the Trans-Blot Turbo Transfer system from Bio-Rad. We use it with our single gel we have made based the One-Gel system by Ahn T et al (2001) (DOI:<a href="https://dx.doi.org/10.1006/abio.2001.5038" target="_blank">10.1006/abio.2001.5038</a>). It should work the same for Laemmli-based gel systems.</p>
<p>We have achieved transfer from proteins ranging from 270 kDa to 26 kDa present in the same gel.</p>
<p>The Turbo Transfer has been standarized in our lab since October 2016, therefore there are no publications yet using the method. Data using the Ahn et al gel system have been used in the following publications.</p>
<pre>Zeitler AF, Gerrer KH, Haas R, Jiménez-Soto LF. Optimized semi-quantitative blot analysis in infection assays using the Stain-Free technology. J Microbiol Methods. 2016 Jul;126:38-41. doi: 10.1016/j.mimet.2016.04.016. PubMed PMID: 27150675.<br />Jiménez-Soto LF, Haas R. The CagA toxin of Helicobacter pylori: abundant production but relatively low amount translocated. Sci Rep. 2016 Mar 17;6:23227.doi: 10.1038/srep23227. PubMed PMID: 26983895; PubMed Central PMCID: PMC4794710.Jiménez-Soto LF, Clausen S, Sprenger A, Ertl C, Haas R. Dynamics of the Cag-type IV secretion system of Helicobacter pylori as studied by bacterial co-infections. Cell Microbiol. 2013 Nov;15(11):1924-37. doi: 10.1111/cmi.12166. PubMed PMID: 23844976.</pre>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Miltenyi Buffer
AUTHORS: Marco Cosentino, Elisa Storelli, Alessandra Luini, Emanuela Rasini, Massimiliano Legnaro, Marco Ferrari, Franca Marino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This recipe is used in the following protocol:</div><div class = "text-block"><span style = "font-weight:bold;">Magnetic bead-based TREG-TEFF cell isolation from PBMC with Miltenyi CD4+CD25+ Regulatory T cell Isolation Kit</span></div></div>

[STEPS]
TITLE: PD-L1 expression as predictor of immunotherapy eligibility in penile squamous cell carcinoma patients
AUTHORS: Muhammad Haritsyah Warli, Fauriski Febrian Prapiska, Ginanda Putra Siregar, Bungaran Sihombing, Syah Mirsya Warli

[DESCRIPTION]
We investigated the correlation between PD-L1 expression and poor prognosis. We examined the correlation between PD-L1 expression and survival of penile SqCC and analyzed the adjusted hazard ratios (HR) of the patients with PD-L1 expression compared to anatomical stage of the tumor.

[STEPS]
SECTION: PD-L1 Antibody Preparation
1. Reagent used to stain PD-L1 in this IHC staining was MD21R clone (Medaysis CA), ready to use (rabbit PD-L1 antibody)

SECTION: Pretreatment
3. Pretreatment was done with EDTA pH8.0, 15 minutes using a Pressure Cooker

15 min 
OR

60 min 95 °C to 99 °C 

7. The detection was done by using post primary procedure for 8 minutes, Bond Wash Solution for two times, and polymer for eight minutes. We used DAB mixture for staining and hematoxylin for counterstaining.

8 min post primary procedure
Bond Wash Solution Two times 
8 min polymer 


SECTION: PD-L1 and Sample Mixing and Detection
6. We mixed the solution with PD-L1 (Medaysis) primary antibody for 1 hour.       
SECTION: PD-L1 Antibody Preparation
2. PD-L1 antibody is diluted with ratio 1 uL PD-L1: 100 uL IHC diluent and then mixed for 5 minutes.



SECTION: Pretreatment
4. Deparaffination and rehydration were done by using Bond Dewax Solution for 3 times, alcohol for 3 times, and Bond Wash Solution for 3 times.

SECTION: Pretreatment
5. We retrieved the antigen by using Bond Epitope Retrieval Solution I for 4 times
SECTION: Analysis
8. PD-LI expression was assessed on cytoplasm and/or tumor cell membranes and TILs. The positive control used was the placenta.
SECTION: Analysis
9. PD-LI expression is considered positive if it has a score of +2 or +3 and is considered negative if it has a score of +1 and 0. Intensity measurement then given a score of 0-3 (0= none; 1= weak; 2= moderate; and 3= strong).
SECTION: Analysis
10. The percentage of stained cells was assessed on a scale of 0-100%. 
Percentage of stained tumor cells A= percentage of tumor cells intensity in 3+ {3 x (%Cells 3+)}, B= percentage of tumor cells intensity in 2+ {2 x (%Cells 2+)}, C= percentage of tumor cells intensity in 1+ {1 x (%Cells 1+)}
SECTION: Analysis
11. Semi-quantitative assessment refers to research by Chovanec et al. who used histoscore (H-score)

H-Score = {1 x (%cells 1+) + 2x (%cells 2+) + 3x (%cells 3+)}
SECTION: Analysis
12. Total value obtained ranges from 0 to 300, thus PD-L1 expression is categorized into “1” for negative (0 – 99) and “2” for positive (100 – 300).
TITLE: Create Mask for Axonal Quantification Analysis with FIJI
AUTHORS: Oscar Moreno Ramos, Elizabeth P.

[DESCRIPTION]
This is a protocol describing how to create a quantified mask from an image of fluorescence-tagged axonal projections using FIJI/ImageJ software.

[STEPS]
1. Upload TIFF image of single-channel fluorescent axons to Fiji
2. Adjust brightness/contrast (keyboard shortcut: ctrl + shift + C)
3. Select Plugins -> Analyze -> Tubeness
4. Enter 0.5 for the sigma value
5. Synchronize windows: select Analyze -> Tools -> Synchronize windows
6. Open thresholding (shortcut: ctrl + shift + T)
7. Adjust the thresholding value to your needs
8. Select apply and create mask.
9. Save created mask as tiff file.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The protocol can be used to create and extrude phospholipid liposomes. This extrusion protocol roughly follows <a href="https://avantilipids.com/divisions/equipment/mini-extruder-extrusion-technique/" target="_blank">some guidelines published by the manufacturer</a>. However, we found that the manufacturer's directions were insufficient in several areas. In particular, the manufacturer guidance on initial preparation of lipids was incomplete; 'good' liposomes cannot be prepared directly from dry lipid mixtures. In addition, our protocol provides supplementary guidance for extrusion and suspension of different classes of polar lipids; efficacy of the various matrixes we recommend was largely determined through trial and error. The manufacturer directions (downloaded March 4, 2017) are attached to this protocol as a .pdf document.</p>
<p> </p>
<p>This protocol was original created by <a href="&quot;http://www.whoi.edu/sbl/liteSite.do?litesite" target="_blank">Krista Longnecker</a> and <a href="http://jamesrco.github.io/" target="_blank">Jamie Collins</a> for creating lipsomes to be used in lipid photo-oxidation experiments. The results of these experiments are detailed in Chapter 4 of:</p>
<p> </p>
<p>Collins, J. R. 2017. The remineralization of marine organic matter by diverse biological and abiotic processes. Ph.D. thesis. Cambridge, Massachusetts: Massachusetts Institute of Technology, 300 pp; doi:<a href="http://dx.doi.org/10.1575/1912/8721" target="_blank">10.1575/1912/8721</a></p>
<p> </p>
<p>A manuscript of the chapter is forthcoming.</p>

[BEFORE_START]
<p><strong>Note: </strong>Plan to use the liposomes you are making within a day or two because they are not stable for longer periods of time. If necessary, store the extuded liposome solutions at 4ºC.</p>
<p> </p>
<p>This protocol assumes you have an <a href="https://avantilipids.com/divisions/equipment/" target="_blank">Avanti mini-extruder with heating block</a>, available at <a href="https://avantilipids.com/product/610000/" target="_blank">https://avantilipids.com/product/610000/</a></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Vandy - Tail-Cuff Blood Pressure
AUTHORS: Chee Lim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">The tail-cuff blood pressure measurement is a non-invasive test and the principle is the same as that of the inflatable blood pressure cuff used in the standard clinic visit. The Visitech system uses an LED light source to detect the pulse signal wave. Since the measurement is sensitive to motion artifact, the procedure requires training sessions to acclimate the mouse to the process. </div></div>

[STEPS]
?. Turn on the BP-2000 computer and control unit and set the heating platform at approximately 34ºC.
?. Place a mouse in the magnetic restraining device on the platform leaving its tail free.
?. Insert and slide the mouse tail all the way through the corresponding tail-cuff and tape the tail down.
?. Place the LED light sensor cover over the middle of the tail.
?. Open the Blood Pressure Analysis software on the computer and set Preliminary Cycles at 5 and Measurement Cycles at 10.
?. During the analysis cycle the blood pressure control unit will pressurize and attempt to read a pulse and begin cycles of pressurizing and depressurizing.
?. Results will be presented at the end of the measurement cycle.
?. Training is repeated in conjunction with data acquisition, with repetition until experimental variability is minimal (typically 3 training sessions)