eamag/protocols-with-steps huggingface dataset:
TITLE: Conventional fixation method for Tetrahymena  thermophila
AUTHORS: miao.tian  

[DESCRIPTION]
A rapid and robust method for fixing Tetrahymena thermophila cells

[STEPS]
SECTION: General description of the method
1. It works for the IF of cytoplasm, nucleoplasm, and chromatin-bound proteins, FISH staining of receptive sequences (e.g., telomeric repeats, TEs). Nuclear and cell morphology are nicely maintained.

It does not work for the IF of Cna1, γ-H2A.X, and tubulin.
SECTION: Reagents
2. -    37% Formaldehyde (! Toxic, handel it in a fume hood)
-    Triton X-100
-    Sucrose
-    Paraformaldehyde (!Toxic; Cat# A3813, 1000. Applichem)
SECTION: Recipes for making solutions
3. Preparing 4% formaldehyde/ 3.4% sucrose fixative 100 mL

SECTION: Recipes for making solutions
3.1. Weight 4 g of paraformaldehyde, add 100 mL of water.

-    Heat to dissolve paraformaldehyde in a fume hood. Namely, placing the flask on top of a 170 °C heat plate with stirring function, for around 25 min, with stirring. Try not let it boil.
-    Leave it on bench to cool for around 30 min (do not make it completely cool, otherwise sugar dissolves very slow).

SECTION: Recipes for making solutions
3.2. Add 3.4 g of Sugar into the warm paraformaldehyde solution, stir/dissolve it.

-    leave it at room temperature overnight and then store it in a fridge.
SECTION: Recipes for making solutions
4. 10% Triton X-100 50 mL
-    Add 5 mL of Triton X-100 into 45 mL of water. Microwave for 5s will let Triton X-100 dissolve rapidly.

SECTION: Fixation of 5 mL of Tetrahymena cells
5. (!) For growing cells: Wash cells once with 10 millimolar (mM) Tris-HCl (pH 7.5) and resuspend cells with the same volume of 10 millimolar (mM) Tris-HCl (pH 7.5). Starvation or conjugation cells need not to be washed for another time.

SECTION: Fixation of 5 mL of Tetrahymena cells
6. Shoot 250 µL of 10% Triton X-100 (first) and 500 µL of 37% formaldehyde into cells. Invert tube ~ 5 times and keep it at room temperature (25 °C) for 30 min;

SECTION: Fixation of 5 mL of Tetrahymena cells
7. Collect cells by centrifugation at 1500 rpm for 1 min and pour supernatant (! contains formaldehyde, think about waste disposal rules)

SECTION: Fixation of 5 mL of Tetrahymena cells
8. Resuspend cell pellet with ~ 500 µL of 4% formaldehyde/ 3.4% sucrose fixative, mixing cells by gentle pipetting is recommended.

SECTION: Fixation of 5 mL of Tetrahymena cells
9. Cells are ready to apply onto slides. After drying in a fume hood for 1hr, slides are ready for staining.
TITLE: Zebrafish 2021 Environmental Summary, Reptile &amp; Aquatics, Stowers Institute for Medical Research
AUTHORS: Diana P Baumann, Adam  Petrie

[DESCRIPTION]
Zebrafish are an established model organism in biomedical research, and we have maintained these fish at the Stowers Institute for Medical Research since 2006. The housing systems and facility rooms allow for standardization of husbandry and environmental parameters. Acknowledging that husbandry conditions and environmental parameters can impact research results we provide information about the housing, nutrition, maintenance, and health for our zebrafish colonies. This information will be applicable to any zebrafish housed in the facility in 2021.

[STEPS]
TITLE: Data Processing and Preparation of MALDI IMS data
AUTHORS: Nathan Heath Patterson, Elizabeth Neumann, Jamie Allen, Danielle Gutierrez, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">How to process a mass spectrometry imaging data into a final imzML file.</div></div>

[STEPS]
?. For each spectrum in the data set, apply total ion current normalization by dividing the intensity values in the spectrum by the sum of the spectrum's intensity values.
?. Generate a mean spectrum for the dataset by taking the mean of each mass bin of the dataset.
?. Internally recalibrate the mean spectrum using well-known, spectrally ubiqitious lipid species, retaining the calibration equation.
?. Extract peak intensity data for every pixel using the provided identified mass list generated here (Lipid Annotation of MALDI IMS Datasets) by inverting the calibration coefficients to get the mass values in the original calibration and pulling intensity values from the Bruker .sqlite file that match these mass values.
?. Process the extracted peak list into a data table of columns: x, y, m/z 1, m/z 2, m/z 3... where 1, 2, and 3 are placeholders for the identified lipid species.
?. Read this table into R and create a Cardinal  MSImagingExperiment object from the pixel coordinates and peak intensity values.https://cardinalmsi.org
?. Use Cardinal's writeImzML() function to write the MSImagingExperiment object to an imzML file.
TITLE: HuBMAP TMC-Florida/Zurich Light Sheet Fluorescence Microscopy Modality Overview
AUTHORS: Jerelyn Nick, Marda Jorgensen, Seth Currlin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is an overview of all of  the protocols currently in use for the Light Sheet Fluorescence Microscopy modality at HubMAP Tissue Mapping Center,TMC-Florida/Zurich.  It includes protocols.io links to each of the individual protocols that make up this project workflow, from processing tissue and  imaging the samples, to uploading the final data to the HuBMAP HIVE.</div></div>

[STEPS]
?. Process donor organs into samples for analysis. Lymph Node: dx.doi.org/10.17504/protocols.io.bbgnijveThymus: dx.doi.org/10.17504/protocols.io.bbgmiju6Spleen: dx.doi.org/10.17504/protocols.io.bc3kiykw
?. Register organ donor, organs received and common coordinate region information in the HuBMAP UUID generator at https://uuid.hubmapconsortium.org.
?. Clear tissue using Clarity protocol:https://www.protocols.io/view/tissue-clearing-using-clarity-method-bppimmke
?. Immunostaining of cleared tissue (large volume): https://www.protocols.io/view/large-volume-immunostaining-for-cleared-samples-bprtmm6n
?. Image acquistion using Light Sheet Fluorescence Microsopy:https://www.protocols.io/view/light-sheet-fluorescence-microscopy-image-acquisit-bprsmm6e
TITLE: Physical property-Stability and pH (TBE and Borax Agarose electrophoresis buffer)
AUTHORS: Stephane  Fadanka, Nadine          Mowoh, Shalo  Minette

[DESCRIPTION]
The pH check is important because changes in the pH might affect mobility of DNA in the agarose gel hence PCR result alteration and misinterpretation.
The physical stability of the electrophoresis buffer is important because if the powder absorbs moisture from air overtime, they might not be very efficient in allowing migration of DNA samples in the gel which could affect the PCR results.
This protocol describes how to assess the physical property of TBE and Borax electrophoresis buffers.

[BEFORE_START]
Ensure all preliminary steps involved in the process are performed (e.g initially weighing and drying buffer powders)

[GUIDELINES]
Quality control tests are important to ensure the reagents are functioning properly. In this procedure we describe how to assess the physical stability and pH of Agarose electrophoresis buffer as this might affect the migration of DNA sample in an Agarose gel electrophoresis.

[STEPS]
SECTION: Confirming Stability and pH
1. 
 

To confirm the weight of the TBE powder buffer sachet:

Weigh the individual buffer components that would make 500 mL of the 10x buffer as indicated in the table below (the individual powders could be pre-dried if necessary in an incubator with silica gel beads at 37 °C).

 

Put them together in a beaker and mix.
Pour the powder mix in a zip lock sachet and store in an air tight container for 1 to 3 days.
After, open the sachets and pour the powder into a weighing boat and measure the weight of the dried powder.
The weight should be approximately close to or equal to the weight of the powder before the 3 days storage.

To confirm the pH of the buffer solution:

Pour the powder buffer into an appropriate size beaker and add the appropriate amount of distilled water as indicated on the powder sachet (e.g 1 sachet in 500 mL distilled water).
Mix until a clear solution is obtained
Use a pH meter to check the pH of the solution which should be between 8.3 and 8.5.
TITLE: Golden Gate Assembly
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to assemble DNA oligos containing Golden Gate restriction sites with a plasmid backbone that contains the same restriction sites. The restriction enzymes utilised in this protocol are the Type IIS restriction enzymes, specifically BsaI. The use of Type IIS restriction enzymes ensures that there is no scar or extra sequence at the junctions between the assembled fragments. 

[GUIDELINES]
This protocol outlines the Golden Gate procedures with a sample volume of 20 µL per reaction.

[STEPS]
SECTION: Golden Gate Assembly
3. Add the following reagents into a PCR tube:

 

 

SECTION: Golden Gate Assembly
1. Prepare an ice box.
SECTION: Golden Gate Assembly
4. Put the sample into the Thermal Cycler and run it with the following conditions: 

*Set "Lid Temperature" to 105 °C and set "Volume" to 20 µL 
 

SECTION: Golden Gate Assembly
2. Place the  ,  , and   in ice.

SECTION: Transformation
5. Prepare a box of ice.
SECTION: Transformation
6. Take an Eppendorf tube that contains pre-made competent cells from the -80 °C fridge.

SECTION: Transformation
7. Immediately place the Eppendorf tube with competent cells into the ice box for 5 min .

SECTION: Transformation
8. Add the whole Golden Gate Assembly product (20 µL) o into the Eppendorf tube containing the competent cells.

SECTION: Transformation
9. Tap the bottom of the Eppendorf tube to mix the solution.
SECTION: Transformation
10. Leave the Eppendorf tube in ice for 10 min .

SECTION: Transformation
11. Place the Eppendoft tube into a foam floating.
SECTION: Transformation
12. Place them into the water bath for 45 s at 42 °C for heat shock.

SECTION: Transformation
13. Place the Eppendorf tube into the ice immediately
SECTION: Transformation
14. Add 1 mL of the LB media into the Eppendorf tube.

SECTION: Transformation
15. Place the Eppendoft tube into the incubator at 37 °C for 60 min for recovery.

SECTION: Transformation
16. Centrifuge the Eppendorf tube to form a cell pellet (no specific speed and time).
SECTION: Plating and Incubation
17. Prepare an LB agar plate with the correct antibiotics.
SECTION: Plating and Incubation
18. Remove 950 µL of the LB solution from the Eppendorf tube that contains the cell pellet, leaving about 100 µL in the Eppendorf tube.

SECTION: Plating and Incubation
19. Resuspend the cells by pipetting the solution.
SECTION: Plating and Incubation
20. Spread the cells onto the agar with the L-spreader.
SECTION: Plating and Incubation
21. Place the petri dish in the incubator at 37 °C for 60 min to allow the colonies to grow.
TITLE: PFA treatment of OP50
AUTHORS: Bonnie Evans

[DESCRIPTION]
Paraformaldehyde (PFA), a polymer of formaldehyde, is an organic solution that permeabilizes bacterial cells making them no longer viable/metabolically active. However, PFA does not cause the lysis of inner cell structure, meaning the bacteria are still edible for the worms. Adapted from Beydoun et al. 2021. 

[STEPS]
SECTION: PFA treatment
5. Follow steps 1 to 12
 

SECTION: PFA treatment
6. In the fume hood, add 16% PFA stock to the culture to give a 0.5% concentration

 

SECTION: PFA treatment
7. Incubate culture for 2 hours at 37C 200rpm 
SECTION: PFA treatment
8. Divide culture into an even number of 50mL Falcons with equal volume 

 

SECTION: PFA treatment
9. Centrifuge for 20 minutes at 4000 rcf 4C
SECTION: PFA treatment
10. Pour off supernatant into a waste flask
 

SECTION: PFA treatment
11. Using a stripette, replace with an equal volume of M9 and re-suspend pellet


SECTION: PFA treatment
12. Repeat steps 6 to 8 twice more
SECTION: PFA treatment
13. Vortex Falcons to fully re-suspend pellet and combine solutions in a larger container (should be the same total volume as the original culture). 
SECTION: PFA treatment
15. Using an inoculation loop, streak the PFA-treated culture on LB agar and incubate overnight at 37C to check for contamination/live bacteria
SECTION: PFA treatment
17. Once PFA killing has been confirmed as successful, aliquot into 50mL Falcons and store at 4C
 

SECTION: PFA treatment
16. Store PFA culture at 4C
SECTION: PFA solution
1. In the fume hood, break off the head of the ampoule of 16% paraformaldehyde (PFA)
SECTION: PFA solution
2. Using a plastic Pasteur pipette, decant the solution into a brown glass bottle. Dispose of Pasteur pipette in Biobin in fume hood. 
SECTION: PFA solution
3. Leave the ampoule on the plastic tray in the fume hood to allow any remaining PFA to evaporate. Dispose of in sharps bin.
SECTION: PFA solution
4. Store the PFA stock solution double contained at 4°C 
SECTION: PFA treatment
14. Measure OD600 using M9 as a blank
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Proteins from the spinal ligament cells of wild-type and null mice were separated using two-dimensional gel electrophoresis and stained with Pro-Q Diamond phosphoprotein gel stain (Thermo Fisher Scientific, Waltham, MA, USA) followed by SYPRO Ruby protein gel stain (Thermo Fisher Scientific). The gel was visualized by using a Molecular Imager FX Pro Plus multi imager system (Bio-Rad, Hercules, CA, USA), and the images were acquired using PDQuest software, version 8.0 (Bio-Rad). The composite images were digitally pseudo-colored and overlaid. The gels were treated with a phosphorylation reagent, and the phosphorylation spots were verified using LC–MS/MS after in-gel digestion using trypsin and peptide extraction, which were performed according to a previously published protocol. Purified peptides (20–30 pmol) were analyzed by using the UltiMate 3000 RSLCnano system (Thermo Fisher Scientific) coupled to an Orbitrap Elite linear ion trap mass spectrometer (Thermo Fisher Scientific) with an in-house manufactured nano-electrospray ionization interface. For micro reversed-phase LC-MS/MS analysis, the samples were injected into a trap column (nano; 75 × 280 μm inner × outer diameter; packed with 15 cm Acclaim Pep Map C18). Buffer A (0.1 % formamide) and Buffer B (80 % acetonitrile and 0.0 8% formic acid) were used to elute the bound peptides with a split flow system (flow rate: 300 nL/min) for 60 min on a linear gradient. In positive ion mode, spectra were acquired with cycles of one full MS scan in the linear trap quadrupole (<em>m</em>/<em>z</em> 350–2000) followed by 20 data-dependent MS/MS scans with a normalized collision energy of 35 %.</p>

[STEPS]
TITLE: Chapter 6: Paralysis
AUTHORS: Kerri Wolter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Paralysis is commonly associated with traumatic injuries and poisoning (carbamate, organophosphate and lead) cases. </div><div class = "text-block">In respect of trauma – these are typically birds which have collided with a power line cable, crash-landed, and impacted the ground with their chest. This tends to result in a compression fracture of the lumbar (back) vertebrae, often resulting in paralysis of the legs, whilst the wings remain functional. </div></div>

[STEPS]
?. [Assessment of paralysis cases]
The most important prognostic test is to check for a pain response. Use a pair of forceps to pinch a toe or skin on the leg.
The bird may well pull its leg up in an involuntary (automatic) response; this is called a spinal reflex. Whilst worth noting, this response only tells us about the nerve supply from the leg to the corresponding spinal segment and back.
?. [Assessment of paralysis cases]
Watch the head for any response. The bird should move its head, usually turning towards the stimulus, perhaps appear alarmed or try to defend itself. These are positive indications that the bird can consciously feel; that nerve impulses have successfully travelled up the spine.
What we are interested in is the connection between the spinal cord and the brain. In order to assess this, we are looking for ‘deep pain response’, or conscious acknowledgement of the pinch.
?. [Assessment of paralysis cases]
A lack of ‘deep pain response’ suggests severe spinal damage and carries a grave prognosis. In these instances, if the bird is not in too much distress, try treating the bird with fluid therapy and NSAIDs, with or without additional analgesia for a maximum of 48 hours.
?. [Assessment of paralysis cases]
If the deep pain response has not returned after 48 hours, euthanise the bird.
?. [Assessment of paralysis cases]
If there is some indication of a deep pain response, initiate supportive care.
?. [Assessment of paralysis cases]
Physical – sling the bird (Fig. 10), providing a poop hole. Always keep the vent clear to allow the bird to defecate clear of the sling to avoid soiling itself.
?. [Assessment of paralysis cases]
Administer fluid therapy (see CHAPTER 2).
?. [Assessment of paralysis cases]
Provide nutritional support (force or tube feed at a rate of 2-3% of bodyweight per day).
?. [Assessment of paralysis cases]
Administer anti-inflammatory medication and pain relief (meloxicam, only given following rehydration).
?. [Assessment of paralysis cases]
Administer a Vitamin B complex.
?. [Assessment of paralysis cases]
Perform physiotherapy to prevent cramping and muscle/tendon contracture, including use of a water bath if appropriate
?. [Assessment of paralysis cases]
If there is no response or improvement within 2 weeks, the case is hopeless and the bird should be euthanised.
TITLE: Poliovirus direct detection and nanopore sequencing (DDNS) FAQs
AUTHORS: Alex Shaw, Catherine Troman, Joyce Akello, Erika Bujaki, Manasi Majumdar, Javier Martin, Nick Grassly

[DESCRIPTION]
This short FAQ document summarises the poliovirus direct detection and nanopore sequencing (DDNS) protocol, the equipment and reagents required, the cost and staff-time involved (v1.7 updated 4 Sep 2023).

[STEPS]
TITLE: Laurel Wilt / Raffaelea lauricola screening from wood pieces with QIAGEN – QIAamp Fast DNA Stool Mini Kit
AUTHORS: Romina Gazis-Seregina, Pedro Pablo Parra

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes DNA extraction of wood samples for the detection of R. lauricola.</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: No Title
AUTHORS: 

[DESCRIPTION]
This is a protocol from: <br /><br />Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_057.pdf" target="_blank">published manuscript</a> for additional information.

[GUIDELINES]
<strong>Authors: </strong>Kenneth M. Stedman<sup>1</sup>, Kate Porter<sup>2</sup>, and Mike L. Dyall-Smith<sup>3</sup> <br /><sup>1</sup>Department of Biology, Center for Life in Extreme Environments, Portland State University, P.O. Box 751, Portland, OR 97207, USA <br /><sup>2</sup>Biota Holdings Limited, 10/585 Blackburn Road, Notting Hill Victoria 3168, Australia <br /><sup>3</sup>Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany<strong><br /></strong><em><br /></em>Several haloviruses have been isolated from laboratory strains of haloarchaea. Most were inadvertent discoveries, based on the spontaneous lysis of the host culture (e.g., ΦH, ΦCh1), or the detection of virus particles in purified preparations of flagella (Hs1). A more systematic approach would be to use induction by mitomycin C, and then to plaque cell supernatants on related (nonlysogenic) host strains. Indeed, this has been recently used to isolate a new halovirus, SNJ1 (Mei et al. 2007), from a strain of Natrinema, and could be used more widely.<em><br /><br /></em><em><br /></em>

[STEPS]
TITLE: Zielh neelsen for coccidia Agudelo y Montoya, 2004
AUTHORS: Javier Antonio Benavides

[STEPS]
?. Place a drop of 0.85% saline on a new, fat-free, dry slide.
?. Thoroughly homogenize the stool sample to be examined with a wooden toothpick
?. Place the wooden toothpick impregnated with the stool sample in the drop of saline solution that was put on the slide and homogenize in such a way that it has a spread of approximately 2 cm long by 2 cm wide
?. Allow drying at room temperature
?. Fix the plate by immersing it in absolute methanol for 5 minutes
?. Let the environment dry
?. Cover the plate with carbol-fuchsine for 20 minutes
?. Remove excess colorant with tap water
?. Discolor with 7% H2SO4 until the plate is pale pink
?. Remove excess colorant with tap water
?. Add methylene blue or malachite green throughout the spread for 3 minutes
?. Remove excess colorant with tap water
?. Let the environment dry
?. Visualize the plate with immersion oil with the objective of 100x
?. Basic fuchsine: Fuchsine 10g diluted in Ethanol 100ml Phenol solution 5% (phenol 5ml add 95ml of water)From mother solution (Basic fuchsine filtered 10ml add 100 ml of phenol solution
?. Methylene blue   10g diluted in Ethanol 95%  100mlFrom the mother solution filter 30ml and add 70ml water
?. Prepare sulphuric acid solution 7% (H2S04)Sulphuric acid 7ml plus Ethanol 93ml
?. Malachite Green solution (Malachite green 5g diluted in Ethanol 10% 100ml)
TITLE: Functional Magnetic Resonance Imaging Under Swallowing Task in Healthy Human Subjects and Gastroparetic Patients
AUTHORS: Kun-Han Lu, Kristine Mosier, John Wo, Terry Powley

[DESCRIPTION]
This protocol described the steps to acquire and analyze functional magnetic resonance imaging data under a swallowing task in healthy human subjects and patients with gastroparesis. 

[STEPS]
SECTION: Subjects
1. Eight healthy subjects (6 females; 2 males) and 3 gastroparetic patients (2 females; 1 male) participated in this study under research protocols approved by the Institutional Review Board at Indiana University School of Medicine. All healthy subjects did not have a prior diagnosis of neurological, psychiatric, or gastrointestinal (GI) disorders. All gastroparetic patients had documented delayed gastric emptying by a standardized 4-h gastric emptying scintigraphy test (Tougas et al., 2000). Written informed consent was obtained from all participants. Standard MRI exclusion criteria were applied. 
SECTION: Experiment design
2. Every subject underwent 3 fMRI sessions. Each subject first had a 5-min, eyes-closed, resting-state fMRI scan. Then, the subject was instructed to perform a 30-sec ON, 30-sec OFF swallowing task during a 5-min fMRI scan. The subject was asked to continuously swallow boluses of water during each 30-sec ON period via a silastic tube and stop swallowing during the 30-sec OFF period. After the swallowing fMRI scan, the subject had another 5-min, eyes-closed, resting-state fMRI scan.
SECTION: MRI acquisition
3. T1 and T2-weighted MRI and fMRI data were acquired in a 3T MRI system (Siemens, Magnetom Prisma, Germany) with a 64-channel receive-only phased-array head/neck coil. The fMRI data were acquired with 2 mm isotropic spatial resolution and 1s temporal resolution by using a gradient-recalled echo-planar imaging sequence (multiband = 8, 72 interleaved axial slices, TR = 1000 ms, TE = 31 ms, flip angle = 52°, field-of-view = 21 cm × 21 cm). MRI/fMRI images were preprocessed using a similar pipeline as in the Human Connectome Project (Glasser et al., 2013). Briefly, all fMRI images were corrected for motion and slice timing, aligned to structural images, and normalized to the Montreal Neurological Institute (MNI) space.