eamag/protocols-with-steps huggingface dataset:
TITLE: Overall protocol for top-down LC-MS/MS of human small intestine tissue
AUTHORS: Jeannie  Camarillo, Bryon  Drown, Neil  Kelleher

[DESCRIPTION]
Overview description of the process for acquiring top-down LC-MS/MS data on human small intestine tissue

[STEPS]
SECTION: Tissue Collection
1. Small intestine tissue was provided by Stanford University TMC. Tissue was collected according to the following: 

https://dx.doi.org/10.17504/protocols.io.bin8kdhw
SECTION: Sample Preparation
2. Perform LC based Proteomics on adjacent tissue sections:

 

SECTION: Data Acquisition
3. 

 


SECTION: Sample Preparation
4.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This work aims for modeling and simulating the metastasis of cancer, via the analogy between the cancer process and the board game Go. In the game of Go, black stones that play first could correspond to a metaphor of the birth, growth, and metastasis of cancer. Moreover, playing white stones on the second turn could correspond the inhibition of cancer invasion. Mathematical modeling and algorithmic simulation of Go may, therefore, benefit the efforts to deploy therapies to surpass cancer illness by providing insight into the cellular growth and expansion over a tissue area. We use the Ising Hamiltonian, that models the energy exchange in interacting particles, for modeling the cancer dynamics. Parameters in the energy function refer the biochemical elements that induce cancer birth, growth, and metastasis; as well as the biochemical immune system process of defense. </p>

[BEFORE_START]
<p>From http://delta.cs.cinvestav.mx/~matias/CancerModelingSimulation/principal.html, take the executable GoIsingCancer. Use the Java platform of NetBeans for run the application. </p>

[GUIDELINES]
<p>Run GoIsingCancer with the SGF (smart game files) input, one per Go/cancer-IS (immune system) game. This is the Go gaming as a metaphor of cancer versus the immune system reaction.</p>
<p> </p>
<p>The output gives the options for: </p>
<p> </p>
<p>1) Display the board and play, step by step, the SGF sequence of Go/cancer-IS game.</p>
<p> </p>
<p>2) Display the CFG from the Go-cancer game step by step. </p>
<p> </p>
<p>3) Display the graph that illustrates, step by step, the Ising energy for each set of Go/cancer-IS stones-cells during a game. </p>
<p> </p>
<p>In addition, you may see: </p>
<p> </p>
<p>1) The given graphical files showing the evolution of the Ising energy from simulations made with SGF files that correspond to Go-cancer and the of the immune system reaction games.</p>
<p> </p>
<p>2) The videos that show the evolution from the SGF of Go/cancer-IS.</p>

[STEPS]
TITLE: A simple laboratory rearing method for chalcid wasps
AUTHORS: Lilian Matallana Ramirez, Kelly Goode, Nicholas Moore, Robert Jetton, John Frampton

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>For several years, studies on insect behavior, larval development, infestation patterns, parasitism, and identification of species have been possible by the implementations of methods and equipment to rear insects in controlled environments. Rearing methods vary from being simple duplication of nature, such as holding insects and their feed resources to those where years of practice are required to tuning the optimal conditions. The vast majority of the rearing protocols are derived empirically from experimenting with a variety of materials and procedures until success is achieved. Because the determination of infestation caused by </span><span style = "font-style:italic;">Megastigmus specularis, a seed-parasite, </span><span>on Fraser fir seeds and their relation to its parasitoid </span><span style = "font-style:italic;">Mesopolobus,</span><span> are fundamental to biological control and related fields, accurate data are of great importance. Our method was established to obtain chalcid alive, using an economic and fast setting-up. This method has been used in our lab for different studies related to chalcids and their parasitoids. We present a very simple method to rear these species</span><span style = "font-style:italic;"> </span><span>under laboratory conditions. The protocol can be used in experiments in which variation in day length and temperature is required. The rearing mounting can be easily placed in any laboratory or inside of a more controlled environment chamber and use inexpensive materials. This rearing method facilitates the observation and capture of adult insects while maximizing the number of experimental units (EUs) that could be evaluated. </span></div></div>

[STEPS]
?. [Rearing protocol]
Holes were punctured into 10 cm x 14 cm clear, plastic, sealable bags for air exchange using dissecting needles.Place approximately 80-100 Fraser fir seeds in each bag. This will be referred to as an experimental unit (EU).Soil media or substrate to the bags can be added to simulate natural conditions. Note: M. specularis and its parasitoid Mesopolobus do not require media in the bags to emerge. Display at constant temperature (CT) and natural photoperiod (NP):EUs are hanged in a metal rack using small binder clips. The rack is located in a room at room temperature (22±2 C°) next to a window at 11 cm under (1-2) T5-54W fluorescent lamps. The lamps are connected to a timer and adjusted to turn on and off following natural photoperiod (NP). These means timers turn on when the sun rises outside and off when it set outside. Display in cold (C) and darkness (D)EUs are hanged in a metal rack using small binder clips. The rack is located in a controlled-environmental chamber at (6±2 C°) and complete darkness. Insect collection Bags were unclipped from the racks for examination at least three times every week to characterize and capture adult insects.
?.
TITLE: Donor Case Selection | HuBMAP | JHU-TMC
AUTHORS: Kyu Sang Han, peihsun.wu, Joel Sunshine, Sashank Reddy, Denis Wirtz

[DESCRIPTION]
Donor case selection protocol for Johns Hopkins TMC describes criteria for human tissue biopsy collection.

[BEFORE_START]
Patient samples are obtained from skin that is to be discarded from patients presenting for reconstructive or aesthetic procedures necessitating skin excision. No other procedures or follow-up is needed for research purposes.

[STEPS]
SECTION: Inclusion
1. Age 18-35 or 50 and over
English Speaking
Able to provide consent
Undergoing reconstructive or aesthetic procedures necessitating skin excision at the scalp or abdomen
SECTION: Exclusion
2. Known collagen disorder
Strong personal history of keloid formation
Deemed unfit for general anesthesia
Active infection of skin where samples may be taken
TITLE: Creating Gelvatol Solution
AUTHORS: Daniela Boassa, Mark Ellisman

[DESCRIPTION]
A gel-like mounting media (with an anti-fade ingredient) for fluorescence slides. 

[BEFORE_START]
 Add a small drop of Gelvatol to specimen.
Carefully place coverslip on the drop(s) avoiding bubbles.
The mounting media will set overnight at 4 degrees or at room temperature 3-6 hours.

[STEPS]
1. Add 2.4 g of Polyvinyl Alcohol (Mol. Wt. 30,000-70,000) to 6ml of glycerol.

2. Stir well to mix.
3. Add 6ml of double distilled water and leave for at least 2 hours at room temperature (rotate overnight)
4. Add 12ml of 0.2M Tris (pH 8.5).

2.4 ml Tris 1M
9.6 ml H2O
5. Heat to 50° C for 10 minutes with occasional mixing. 
6. After polyvinyl alcohol is dissolved, clarify by centrifugation (5000g = ~6800 rpm) for 15 minutes at 20° C.
7. Use the large tabletop 5810R Eppendorf centrifuge.
8. Collect supernatant liquid and measure amount collected. 
9. Add DABCO to 2.5% as antifade medium:

2.5g DABCO / 100ml of liquid
10. pH to 8.6 with pH paper (the paper will disintegrate, so pipette onto paper rather than putting paper into container). 
11. Aliquot in 0.5ml microcentrifuge tubes and store at -20° C. 
12. Stocks of Gelvatol are supposed to be stable at room temperature for several weeks after thawing.
TITLE: Electron microscopy (EM) and quantification
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Electron microscopy (EM) and quantification

[STEPS]
SECTION: Electron microscopy (EM)
1. 
Hippocampal neurons were plated at a density of
60,000 cells/cm2. DIV-3 neurons were infected with lentiviruses
carrying α-syn tagged at the C-terminus to mScarlet, or mScarlet alone
(multiplicity of infection or MOI=2.5). The transduced neurons were cultured to
maturity (DIV17-DIV21) before imaging. The ~100 % infection efficiency of the
mScarlet-tagged constructs was confirmed before using the coverslip for EM
experiments. 

SECTION: Electron microscopy (EM)
2. EM embedding protocol for monolayer cultured cells was performed
on ice. The coverslips were fixed in 2 % glutaraldehyde and in 0.1 M sodium
cacodylate (SC buffer) buffer for at least 60min at 4 °C, washed 3 min with 0.1
M SC buffer (5 times), and postfixed in 1% osmium tetroxide (OsO4) diluted in
0.1 M SC buffer for 30 min to 1hr. 


SECTION: Electron microscopy (EM)
3. The coverslips were then washed for 3 min (5
times) with 0.1 M SC buffer, followed by a water rinse, sequential ethanol
dehydration (20%, 50%, 70%, 90%, 2x100%) 1min each, and subsequent incubation
with dry acetone for 1 min at room temperature (2 times). 
SECTION: Electron microscopy (EM)
4. The samples were then incubated with a Durcupan:Acetone solution (50:50%) for 1hr in rotation,
followed by incubation with 100% Durcupan for 1 hr (2 times). The cells were
then embedded using foil plates along with wood sticks in the embedding oven
(overnight incubation). 
SECTION: Electron microscopy (EM)
5. 60 nm ultrathin sections were cut with a diamond knife
and mounted on 300 mesh grids. Grids were post-stained with 2% of uranyl
acetate for 5 min and lead for 1 min. JEOL 1400 Electron Microscopy and Gatan
digital Camera were used to obtain images. 
SECTION: quantification
6. 



For quantification of inter-vesicular (IV) distances between vesicles, we
considered all the vesicles in the synapse encompassing 1µmx1µm. To mark each
vesicle, a line was drawn across the center of the vesicle, recording its
diameter and its position.                                                        







SECTION: quantification
7. These parameters were then fed into our algorithm on MATLAB (RRID:SCR_001622)( http://www.mathworks.com/products/matlab/), which gave the output of IV distances
for a particular image. The IV distance for all images per condition was pooled
together. This algorithm is available upon request.
TITLE: Collection of skin biopsies from cattle
AUTHORS: Froylan Sosa, Peter Hansen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a simple protocol for collecting skin biopsies from cattle. The procedure described here was used to collect skin for histological analysis but it could also be used to collect skin for PCR or other purposes. Our experience indicates that there are few complications with the procedure. The most common is that occasionally there is a slight amount of bleeding that can be treated by applying pressure using a gauze. Healing occurs rapidly and biopsy sites are difficult to detect 14 days after the procedure.  The protocol is best read by using the attached pdf file. </div></div>

[STEPS]
?. [Procedure]
Restrain the animal in a head chute.
?. [Procedure]
Label the cassettes - make sure to use a permanent marker for histology or pencil.
?. [Procedure]
Clip the biopsy area with clippers and wipe down with ethanol-soaked gauze. We typically sample the thorax (middle of 5 or 6th rib) - or shoulder area
?. [Procedure]
Wipe area with a sterile dry gauze.
?. [Procedure]
Spray 5% lidocaine onto the biopsy area and wait for 30 sec.
?. [Procedure]
Collect skin sample by rotating the 5 mm skin punch while pressing downwards with mild force.
?. [Procedure]
Clean the skin punch by immersing in 70% ethanol. The skin punch can be used three to four times.
?. [Procedure]
Take the skin sample from skin punch with a needle or tweezers, and place it into a cassette between the sponges. Close the cassettes.
?. [Procedure]
Place the cassette into the container with 10% NBF at room temperature.
?. [Procedure]
Place sterile gauze on the biopsy site to remove excess blood or stop bleeding.
?. [Procedure]
Place some super glue into the wound site. Wait 10 seconds.
?. [Procedure]
Spray tick spray around the biopsy site.
?. [Procedure]
To process the biopsy, leave the skin samples in NBF for 16-20 h at room temperature. Rinse the skin-containing cassettes in PBS twice, 15 min each time. Cut the skin samples in half and place them back into the cassette (use gloves). Store cassettes in a container containing PBS.
TITLE: Detection of Equatorial Plasma Bubbles
AUTHORS: Swapna Raghunath

[DESCRIPTION]
Slant Total Electron Content values from Global Navigation Satellite System are used to detect the Equatorial Plasma Bubbles in the Ionosphere.

[STEPS]
1. Download GPS data from SOPAC website in RINEX format
2. Data is converted from RINEX to .cmn format using GPS TEC software
3. Calculate ROT and ROTI
4. Difference between Evening ROTI and Day time ROTI is calculated
5. If the difference exceeds 0.05, then Equatorial Plasma Bubble is Detected
TITLE: Hematoxylin and Eosin Stain of FFPE Tumor Tissue
AUTHORS: John Herndon, William Gillanders

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the steps for doing a manual Hematoxylin and Eosin (H&E) Stain of formalin fixed tissue. This stain is used to distinguish tumor tissue, background normal and stromal tissue, as well as tumor infiltrating immune cells. All pathology reviews of tumor tissue received from study participants is performed by examining the H&E stained tissue.</div></div>

[STEPS]
?. [Tissue Preparation]
De-Paraffization of slidesPlace slides in to oven at 60o C for one hour
?. [Tissue Preparation]
Place slides in to Fresh Xylene for 2 minutes, repeat
?. [Tissue Preparation]
Rehydration of sections100% Ethanol, 1 minute, repeat
?. [Tissue Preparation]
90% Ethanol, 1 minute, repeat
?. [Tissue Preparation]
80% Ethanol, 1 minute, repeat
?. [Tissue Preparation]
70% Ethanol, 1 minute, repeat
?. [Tissue Preparation]
Distilled Water, 2 minutes
?. [Tissue Preparation]
Staining StepsHematoxylin 4 minutes
?. [Tissue Preparation]
Rinse in running Tap water until clear
?. [Tissue Preparation]
Clarifier 1, 30 seconds
?. [Tissue Preparation]
Rinse in tap water for 30 seconds
?. [Tissue Preparation]
Bluing reagent, 1 minute
?. [Tissue Preparation]
Rinse in Tap water for 30 seconds
?. [Tissue Preparation]
95% Ethanol 1 minute
?. [Tissue Preparation]
Eosin 1 minute
?. [Tissue Preparation]
Dehydration steps100% Ethanol 1 minute, repeat
?. [Tissue Preparation]
Xylene, 1 minute
?. [Tissue Preparation]
Mount slides with 20 x 50 mm Coverslips using Cytoseal XYL, making sure that there are no air bubbles beneath the coverslip. Press the coverslip gently to remove an air bubbles that may have formed.
?. [Tissue Preparation]
Let slides dry in a horizontal position in a fume hood for at least 1 hour.Slides can be stored indefinitely at room temperature in a slide box
?. [Tissue Preparation]
For illustration, digital storage and possible pathology review slides are scanned on the Leica Aperio slide scanner. The file output is .svs and can be viewed by downloading freely available software from Leica.
TITLE: Preparing worms for the COPAS (wormsorter)
AUTHORS: Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is a guide of how to prepare the worms to be used in the COPAS (wormsorter)</div></div>

[STEPS]
?. Wash the worms off the plates using few milliliters of M9 and collect in a 15ml falcon tube and fill up till 15ml with M9
?. Centrifuge the tube at 1500rpm for 2mins followed by removal of the supernatant using a pasteur pipette and addition of another 15ml of M9
?. Repeat the steps 2 more times
?. After the final wash remove the supernatant as much as leaving behind 10ml in the falcon tube
?. Consolidate all the worm liquid in a 50ml tube and dilute if necessary, for the COPAS
?. Ideally make 2x50ml Falcon tubes
?. Dispense the worms using the COPAS following the protocol COPAS wormsorter
TITLE: Primer TE (10mM Tris, 0.1mM EDTA, pH8.0)
AUTHORS: Jacquelina.Woods  

[DESCRIPTION]
This method was developed at the FDA’s Center for Food Safety and Applied Nutrition for GenomeTrakr’s pandemic response project, monitoring SARS-CoV-2 variants in wastewater​​. Protocols developed for this project cover wastewater collection, concentration, RNA extraction, RT-qPCR detection, library prep, genome sequencing, quality control checks, and data submission to NCBI. This method provides a reagent formula required in the extraction of RNA from viral concentrates using the RNeasy and Zymo kits. 

[STEPS]
1. Mix components together.
2. Store at Room temperature. 

1.1. 100 µL ,, or equivalent. 

1.2. 20 µL ,, or equivalent. 

1.3. 9.88 mL , or equivalent.
TITLE: Retrospective audit
AUTHORS: Kajiru Gad Kilonzo, Stefanie J. Krauth, Jo Halliday, Clive Kelly, Stefan Siebert, Gloria Temu, Christopher Bunn, Nateiya M Yongolo, Sally Wyke, Emma McIntosh, Blandina Mmbaga

[DESCRIPTION]
This protocol details retrospective audit.

[STEPS]
SECTION: Retrospective audit
1. A retrospective audit of all adults aged 18 years or above of hospital medical records for signs & symptoms related to joint pain and arthritis-related diagnoses and relevant comorbidities will be undertaken. The audit will establish a retrospective frequency of joint pain among patients admitted to the medical wards between 1st January, 2017 and 31st December, 2019.
SECTION: Retrospective audit
2. These reviews will be conducted by trained project personnel and the extracted data will include no personal identifying information. All patients’ records are stored within the medical record department at KCMC.
SECTION: Retrospective audit
3. Trained project personnel will approach a dedicated person at the department to identify and pull out and register all hardcopy and softcopy medical department patients’ admission records for the years 2017 -2019 with exclusion of those below 18 years.
SECTION: Retrospective audit
4. Documentation will be undertaken to show retrieved files, included files, excluded and reasons for exclusion or missing admission records.
SECTION: Retrospective audit
5. Information will be captured and recorded onto a standardised questionnaire within the ODK platform, including a patient's unique number, age, gender, district of residence, level of education, occupation, religion, tribe, main complains, signs and symptoms, diagnosis, investigations done, treatment, duration of hospital stay and number of admissions per year. Outcomes of admission such as death, referral to a higher facility and discharge home will also be collected.
SECTION: Retrospective audit
6. The diagnosis captured will be the final diagnosis at the time of discharge, death or referral. Patients’ re-admissions within the years 2017-2019 will be documented as separate individual admissions but marked and the patient’s unique number will be similar for all years that the individual has been admitted. This is to allow documentation of trends and characteristics of admissions among specific individuals over the years.
SECTION: Retrospective audit
7. Data obtained from this retrospective survey will be compared with community and prospective survey data. This will allow researchers of this study to explore and characterize the hospitals past and present burden of NCD disease while projecting the future situation regarding most NCDs associated with hospital admissions.
SECTION: Retrospective audit
8. All information gathered will be provided to the Tanzania hospital management, community and health policy makers via our dissemination activities including a policy brief.
TITLE: A large scale bacterial attraction assay: a new quantitative bacterial migration assay suitable for genetic screens
AUTHORS: Thomas Quiroz Monnens, Alice Boulanger

[DESCRIPTION]
Bacteria use various motility mechanisms to explore their environments. Chemotaxis is the ability of a motile bacterial cell to direct its movement in response to chemical gradients. A number of methods have been developed and widely used to study chemotactic responses to chemoeffectors including capillary, agar plug, microscopic slide, and microfluidic assays. While valuable, these assays are primarily designed to monitor rapid chemotactic responses to chemoeffectors on a small scale, which poses challenges in collecting large quantities of attracted bacteria. Consequently, these setups are not ideal for experiments like forward genetic screens.  To overcome this limitation, we developed the Large Scale Bacterial Attraction assay (LSBA), which relies on the use of a Nalgene™ Reusable Filter Unit and other materials commonly found in laboratories. We validate the LSBA by investigating chemoeffector kinetics in the setup and by using chemoattractants to quantify the chemotactic response of wild-type, and motility impaired strains of the plant pathogenic bacterium Xanthomonas campestris pv. campestris and the environmental bacterium Shewanella oneidensis. We show that the LSBA establishes a long lasting chemoeffector gradient, that the setup can be used to quantify bacterial migration over time and that the LSBA offers the possibility to collect high numbers of attracted bacteria, making it suitable for genetic screens.

[STEPS]
TITLE: DNA Extraction from Bacteriophages
AUTHORS: Frej   Larsen

[DESCRIPTION]
DNA/RNA extraction is performed using the QIAamp® Viral RNA Mini kit from Qiagen without the addition of carrier RNA to the AVL buffer and an additional nuclease step prior to extraction. The nuclease step is introduced to degrade any DNA/RNA that may be in the sample after the virome extraction protocol (dx.doi.org/10.17504/protocols.io.b2qaqdse) has been performed. Please note that even though the kit is a viral RNA extraction kit, viral DNA will also be extracted.




[GUIDELINES]
Before beginning this protocol, ensure that wash buffers have been properly diluted with ethanol and that the centrifuge is available and not chilled as low temperatures may impede ethanol evaporation.

Note that, unlike when using the kit directly, carrier RNA should not be added to the AVL buffer.

The Pierce Universal nuclease should be diluted 100 times (ie. by mixing 1 uL nuclease with 99 uL SM buffer/sterile water)

Wear gloves when performing this protocol.

[STEPS]
1. For each sample, pipet 1 µL  100x diluted Pierce Universal Nuclease (2.5units/uL) to an empty 1.5 mL tube.

2. Transfer 140 µL of each sample from the outer chamber of the CentrisArt filter tube to the tube and mix by pipetting

3. Incubate for at least  3 min  at room temperature. 

4. Add 540 µL AVL buffer to inactivate nucleases and lyse phage heads. Mix by pipetting or pulse vortexing.

5. Incubate 10 min  at room temperature

8. Add 560 µL  absolute ethanol. Mix thoroughly by pulse vortexing.

10. Transfer 630 µL  of the sample to a spin column

9. Briefly centrifuge samples with a microcentrifuge
11. Centrifuge  6000 x g, 1 min, 21 °C or until all liquid has passed through the filter. 

12. Change the collection tube and    until all of the sample has passed through the filter.

13. Add 500 µL  AW1 wash buffer to each spin column.

14. Centrifuge  6000 x g, 1 min, 21 °C or until all liquid has passed through the filter
15. Add 500 µL  AW2 wash buffer to each spin column.

16. Centrifuge  20000 x g, 3 min, 21 °C or until all liquid has passed through the filter.
17. Change the collection tube, then centrifuge  20000 x g, 1 min, 21 °C  to dry the membrane.
18. Place the spin column in a new RNAse-free 1.5 mL tube. 
20. Incubate at room temperature for at least  1 min . 

21. Centrifuge  6000 x g, 1 min, 21 °C 
22. Discard the spin column and store eluate at -80 °C 

6. Change gloves.
7. Briefly centrifuge samples with a microcentrifuge.
19. Add 30 µL  AVE elution buffer directly onto the filter membrane in the spin column.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a slightly modified version of Thermo Fisher's gateway BP protocol which uses less enzyme and is therefore more economical.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.