eamag/protocols-with-steps huggingface dataset:
TITLE: Sampling and  viral concentration for SARS-CoV-2 detection in wastewater 
AUTHORS: Veronica  Antelo, Gregorio Iraola

[STEPS]
?. [Sampling wastewater]
Autoclave plastic containers and expose to UV for 10 min.
?. [Sampling wastewater]
Fill bottles with wastewater at sampling points using sterile gloves.
Sampling points may be diverse and accessibility to water sources will be sometimes difficult depending on site infrastructure. Consider these aspects before starting sampling.
If site is inaccessible by hand, use a rope tied to the bottle to reach narrow or deep places. When recovering the bottle, immediately tap it and sanitize the outside using EtOH 70%.
?. [Sampling wastewater]
Place bottles in a refrigerated container (i.e. with ice flakes or gel packs) until returning to the lab. Our sampling days usually take 4-6 hours since first sample is collected.
?. [Concentration by ultracentrifugation]
Prepare your reagents and materials:
?. [Concentration by ultracentrifugation]
Place 42 mL of sewage water in an appropiate ultracentrifugation tube and ultracentrifugate as follows:
Centrifuge: 100000 34
4 °C
?. [Concentration by ultracentrifugation]
Discard supernatant resuspend pelleted viral particles as follows:
?. [Concentration by ultracentrifugation]
Discard supernatant.
?. [Concentration by ultracentrifugation]
Resuspend viral particles in .
[glycine buffer]
?. [Concentration by ultracentrifugation]
Incubate on ice for
?. [Concentration by ultracentrifugation]
Add  and gently mix.
[PBS]
?. [Concentration by ultracentrifugation]
Clarify supernatant by centrifugation as follows:
Centrifuge: 12000 34
?. [Concentration by ultracentrifugation]
Finally recover viruses by ultracentrifugation as follows:
Centrifuge: 100000 34
4 °C
?. [Concentration by ultracentrifugation]
Resuspend viruses in .
[PBS]
?. [Concentration by ultracentrifugation]
Process immediately for nucleic acid extraction or store at .
-80 °C
TITLE: Media Prep: 500mL of 1x PBS
AUTHORS: Melanie Palma Avila, George  Testo

[DESCRIPTION]
Phosphate-buffered saline (PBS) is an isotonic solution that is used in many biological research applications.PBS (phosphate buffered saline) is a balanced salt solution used for a variety of cell culture applications, such as washing cells before dissociation, transporting cells or tissue, diluting cells for counting, and preparing reagents. PBS is formulated without calcium and magnesium for rinsing chelators from the culture before cell dissociation.

[GUIDELINES]
Storage conditions: 15°C to 30°C

Shipping conditions: Room temperature

Shelf life: 24 months from date of manufacture

[STEPS]
1. Perform a 1:10 dilution of 10x PBS using a sterile media bottle of 1L volume or greater (if necessary).
2. Place cap directly on top of sterile media bottle (DO NOT tighten cap).
3. Autoclave sterile media with 1x PBS: Liquid cycle: media (30 min).

4. Retrieve autoclaved 1x PBS & fully cap sterile media bottle for storage.
1.1. Combine 450 mL DI H2O and 50 mL 10x PBS into sterile media bottle.
TITLE: TS Procure 812 - primary fixation Karnovsky&#39;s - transwells (TM - 013)
AUTHORS: sandra.crameri

[DESCRIPTION]
The method is used to Karnovsky's EM fixed transwells to Procure 812 resin blocks. The presence of 4% paraformaldehyde is only used to satisfy microsecurity requirements.

[GUIDELINES]
Sample is not optimal for EM, best to dissect and use only the outer 2mm of tissue mass. Sample is usually derived from histology.

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR & STEPS:




OPERATOR & STEPS:
SECTION: Conventional
3. Karnovsky's fixative: 2.5 % volume   and 4 % volume   in 0.1 Molarity (M) Sorenson's Phosphate buffer (pH 7.2, 300mosmol/kg) for at least 40 min  or overnight

SECTION: Conventional
4. 0.1 Molarity (M) buffer 15 min 

SECTION: Conventional
5. 1 % volume   in buffer 60 min 

SECTION: Conventional
6. 70 % volume   for at least 20 min  or overnight

SECTION: Conventional
7. 95 % volume    20 min 

SECTION: Conventional
8. 100 % volume    20 min 

SECTION: Conventional
9. 100 % volume    dried with molecular sieve 20 min 


SECTION: Remove membrane
10. Remove membrane from transwell scaffold using a 45o scalpel and transfer to a glass vial with 100 % volume 
  dried with molecular sieve.

SECTION: Conventional
11. 50 % volume   50 % volume   20 min 

SECTION: Conventional
12. 100 % volume   20 min 

SECTION: Conventional
13. 50 % volume   50 % volume Procure 812


SECTION: Conventional
14. 100 % volume Procure 81220 min 

SECTION: Conventional
15. 100 % volume Procure 812 60 min 

SECTION: Conventional
16. Embed & polymerise overnight in oven at 60 °C , section with diamond knife as usual.

SECTION: Conventional
2. All solutions are exchanged above and below the membrane, up to step 10.
TITLE: Impact of Atrial fibrillation on the outcome of patients with Brugada syndrome:  A meta-analysis
AUTHORS: Max Aboutorabi

[DESCRIPTION]
Background: A systematic review and meta-analysis will be conducted to assess the effects of atrial fibrillation (AF) on patients with brugada syndrome (BrS).


Methods: Studies enrolling patients with BrS and AF will be included. Primary outcomes will include major arrhythmic events, syncope, and all-cause mortality, while secondary outcomes will include the rates of SCN mutations. 


Results: We will search OVID MEDLINE, Cochrane CENTRAL, and EMBASE up to January 2024. Data will be extracted and analysed using standard meta-analytic techniques.


Conclusion: Our findings will provide insights into the effects of AF in patients with BrS, aiding clinical decision-making in this population.

[STEPS]
1. Identify the question
Population - patients with Brugada syndrome with and without AF (No constraints were set in terms of type of atrial fibrillation or type of brugada syndrome).
Comparison -  assessing whether concurrent AF has an effect on patients with brugada syndrome.
Outcomes - Rates of major arrhythmic events, syncope, mortality rate, and SCN mutations.


2. Search strategy
Searches were run via OVID MEDLINE, Embase, and Cochrane central from inception to January 2024.
Search terms will include: Brugada syndrome, atrial fibrillation, ventricular fibrillation, ventricular tachycardia, sudden cardiac death, major arrhythmic event
Two reviewers will independently screen for titles for relevance and then assessed the full texts for their eligibility. 
A third expert will intervene whenever discrepancies are observed.
3. Data Extraction
The data will be extracted onto a pre-made excel spreadsheet (Office 365, Microsoft Corporation) with subheadings for study type, single or multi-centre, study drugs, and population characteristics: age, sex, brugada type, AF type prevalence, major arrhythmic event definition, and follow up period.
4. Data analysis
The data will be analysed using odds ratios as a measure of effect. A random-effects model will be used for accounting for the observed heterogeneity. Statistical analysed and creation of forest plots will be completed using meta packages in R programme and variance will be calculated using paule-mandel model.
5. Risk of bias Assessment
The studies included will be assessed by 2 independent authors and will use the Newcastle Ottawa scale.
TITLE: Food4Gut multicenter randomized placebo-controlled trial.
AUTHORS: Sophie Hiel, Marco A. Gianfrancesco, Julie Rodriguez, Daphnée Portheault, Quentin Leyrolle, Laure B. Bindels, Carolina Gomes da Silveira Cauduro, Maria D.G.H. Mulders, Giorgia Zamariola, Anne-Sophie Azzi, Gaetan Kalala, Barbara D. Pachikian, Camille Amadieu, Audrey M. Neyrinck, Audrey Loumaye, Patrice D. Cani, Nicolas Lanthier, Pierre Trefois, Olivier Klein, Olivier Luminet, Jérôme Bindelle, Nicolas Paquot, Miriam Cnop, Jean-Paul Thissen, Nathalie M. Delzenne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Objectives:We describe the methodology of a trial designed to highlight the interest of vegetables rich in ITF-prebiotics in obese adults.</div><div class = "text-block"><span>DESIGN:150 obese subjects (BMI> 30 kg.m</span><span style = "vertical-align:super;">-2</span><span>; 18–65 years) recruited in three different university hospitals in Belgium will be selected with at least one of the following criteria: (pre-)diabetes, hypertension, dyslipidemia, liver steatosis. They will be randomly assigned to receive either 16 g/day of inulin (Fibruline, Cosucra) with dietary advice to promote the consumption of vegetables rich in ITF or 16 g/day placebo (maltodextrin, Cargill) with dietary advice provided nutrients to promote the consumption of vegetables poor in ITF for 3 months. Review of literature, analysis of ITF content in vegetables and satisfaction and digestive tolerance tests (on healthy volunteers) will be performed to propose adequate receipts.</span></div><div class = "text-block">Results: The primary outcome is to evaluate the impact of a higher consumption of ITF-prebiotics on the gut microbiota composition. The secondary outcome of this trial was the effect of the prebiotic intervention on the BMI. Other secondary endpoints were anthropometric and clinical parameters.</div><div class = "text-block">Conclusions:The goal of this intervention study is to establish the proof of concept that nutrients which selectively stimulate the growth of beneficial bacteria in the human colon might offer protection against metabolic disorders associated to obesity on an adequate target population. </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Gen-IALFirst All-tissue DNA extraction kit -This protocol provides an efficient DNA extraction and purification of fresh sample (tissue material)</p>

[BEFORE_START]
<p>Clean</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Dentitox Pro: Does Dentitox Drops scam or real?
AUTHORS: Litsa  Pro

[DESCRIPTION]
Dentitox Pro: Does Dentitox Drops scam or real?

[STEPS]
SECTION: Dentitox Pro: Does Dentitox Drops scam or real?	 Regularly brushing one&#x27;s teeth won&#x27;t help goo complaint or tooth decay. According to the medical community, indeed persons who seek to maintain excellent oral health by drawing their teeth regularly might succumb to these oral issues. This is due to the absence of introductory dental care in a population with poor habits that continue to rise. These habits include smoking, eating reused foods, and drinking sticky products.  The maturity of dentists and dental conventions just treat the symptoms of an oral infection; these treatments should rather concentrate on eradicating all of the dangerous origins that live deep below the epoxies. They&#x27;re slow- moving cutthroats able of escaping discovery for times.  Dentitox Pro is a brand-new product that promotes oral health and prevents tooth decay. It achieves this by avoiding tooth decay and abrasion and drawing the teeth. Continue reading to get an in- depth understanding of the product&#x27;s goods.  The review describes the ineffectiveness of brushing without this supplement. It kills bacteria and aids in removing stains caused by certain refections, similar as red wine.  What&#x27;s Dentitox Pro?  Dentitox Pro is an each-natural dental health supplement that promotes excellent oral health, prevents goo complaint, and strengthens teeth, hence reducing the threat of oral issues for those who like eating and drinking. The absence of paddings, complements, and preservatives makes the product respectable for pregnant women. It&#x27;s an organic herbal admixture with several benefits, including the creation of healthy epoxies. It&#x27;s composed substantially of peppermint excerpt and helps strengthen teeth.   Official Website:-https://www.outlookindia.com/outlook-spotlight/dentitox-pro-reviews-is-dentitox-pro-drops-scam-or-works--news-203893 https://www.homify.in/projects/1158619/dentitox-pro-does-dentitox-drops-scam-or-real https://dentitox-pro-does-dentitox-drops-scam-or-real.jimdosite.com/ https://educatorpages.com/site/osofo80/pages/dentitox-pro-does-dentitox-drops-scam-or-real https://www.pinterest.com/pin/1145532855214971160/
1.
TITLE: 10x Protocols: Chromium Nuclei Isolation Kit Sample Prep -- University of Minnesota TMCs (CG000505 Rev A)
AUTHORS: 10x Genomics

[DESCRIPTION]
Protocol ID# (CG) and Revision letter provided here: 
    10x CG000505, Revision A -- Nuclei isolation kit by 10x Genomics

Note: These protocols may not be the current version offered by the company but were used to produce the specific datasets connected to them. Please review the company support websites for the most recent versions of the protocols prior to starting your experiment. 

[BEFORE_START]
Note: This may not be the most up to date version, just what was used to process samples attached to this DOI. See 10x Genomics for their most up to date revision status prior to attempting.  

[STEPS]
1. Current Sample prep website: https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/sample-prep/chromium-nuclei-isolation-kit-sample-prep-user-guide
2.
TITLE: Cremophor EL agar (EL slant)
AUTHORS: Amy Gladfelter

[DESCRIPTION]
This media is used to determine the ability of Malassezia species to utilize Cremophor EL.

[GUIDELINES]
This media is used to determine the ability of Malassezia species to utilize Cremophor EL.

[STEPS]
1. SDA 65 g

2. Cremophor EL (Sigma) 10 mL

3. dH2O 900 mL

4. Sterilize by autoclaving.
TITLE: Immunohistochemistry
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Immunohistochemistry

[STEPS]
1. Mice were euthanized using isoflurane (VetOne Cat#502017) inhalation followed by
cervical dislocation. At autopsy, mouse brains were removed from the calvarium
and rapidly placed in a 4% paraformaldehyde (PFA) solution (Thermo Scientific,
Cat# J19943-K2) for drop fixation.
2.  For immunohistochemistry, 5 mm sagittal tissue sections were stained with antibodies against total a-syn (Synaptic Systems, 1:15,000, Cat #128211) and Ser129P a-syn (Cell Signaling, 1:3000, Cat #23706). Slides were stained using Ventana Discovery Ultra (Ventana Medical Systems, Tucson, AZ, USA). 
3. The sections were deparaffinized by three cycles of heating to 72°C in the presence of Discovery Wash (Ventana, Ref# 07311079001) for 4 min with a rinse between each heating step.
Subsequently, antigen retrieval was performed using CC1 buffer (Tris-EDTA; pH
8.6 Ventana, Ref# 06414575001) for 40 min (5 cycles of 8 min each with fresh
CC1 buffer for each incubation) at 100°C. Endogenous peroxidase was quenched by
incubation with inhibitor CM (Ventana, Cat# 760-159) for 8 min. The sections
were incubated with primary antibodies for 32 minutes at 37 °C, mixing the samples every 4 min to
ensure an even distribution of the antibody. 






4. Primary antibodies were detected
using the OmniMap system, with an anti-rabbit secondary antibody coupled to an
HRP polymer (Roche Cat# 760-4311, RRID:AB_2811043) (Ventana, Ref# 05266548001).
Antibody presence was visualized using the Ventana Discovery ChromoMap DAB kit
(Ventana, Ref# 05266645001) as a chromagen followed by hematoxylin II (Ventana,
Ref#05277965001) for 4 min followed by a 4 min treatment with bluing reagent
(Ventana, Ref# 05266769001) as a counterstain. 
5. The slides were rinsed,
dehydrated with alcohol and xylene, and mounted on coverslips. To confirm the
specificity of the pSer129 antibody, the epitope was dephosphorylated using
Lambda Protein Phosphatase (New England Biolab, Cat # P0753). Briefly, 2400
units of the Lambda Protein Phosphatase were supplemented with a 10x NEBuffer
and 10x MnCl2 and added to the slides for 2h at 37°C. Antigen retrieval and peroxidase
quenching were performed on the slides before incubation with the pSer129
antibody. 
6. For quantification of Ser129P staining, after background correction,
the following ROIs were placed to sample the frontal lobe and hippocampus – ROI
measuring ~ 2.5 mm x 1 mm flanking the frontal pole, and an irregular ROI over
the CA3 region (which was strongly positive for Ser129P and easy to identify).
Average intensities were considered for quantification, and data were analyzed
in GraphPad Prism software [(RRID:SCR_002798) http://www.graphpad.com/].
TITLE: Evans blue assay to stain dead cells.
AUTHORS: Santosh Sathe

[DESCRIPTION]
Evans blue (Sigma, USA, catalog no: E2129) is a dye that can enter the compromised cell membranes of dead cells and stain them blue. I used this assay to differentiate dead cells in algal cultures of Chlorella and Chlamydomonas (the same protocol can be used for other green algae). 

[STEPS]
SECTION: Evans blue assay to stain dead cells.
1. Contact: Santosh Sathe (santosh.sathe@uni-konstanz.de) 
SECTION: Introduction
2. Evans blue (Sigma, USA, catalog no: E2129) is a dye that can enter the compromised cell membranes of dead cells and stain them blue. I used this assay to differentiate dead cells in algal cultures of Chlorella and Chlamydomonas (the same protocol can be used for other green algae). 
SECTION: Preparation
3. Prepare 1% Evans blue (w/v in MilliQ water) by dissolving 0.1 gm Evans blue in 10 milliliter MilliQ water (I use Falcon tubes). Filter the dye through 0.2 Micron filters using a syringe and filter assembly (bigger pore size filters can also be used). I filter the dye to remove large undissolved particles of dye. The solution (cover it with aluminium foil) is stable at room temperate for several months. 
SECTION: Assay
4. Take 900 µl algal culture in 1.5 milliliter Eppendorf tube (I avoid 2 milliliter Eppendorf tubes as in these tubes the cells do not pellet properly; see below). If the algal cultures are less dense (less than a million cells/ milliliter), then a few milliliter of culture can be centrifuged, and the cell pellet combined and suspended in 900 µl culture media.
SECTION: Assay
5. Add 100 ul Evans blue (from 1% dye suspension prepared above).
SECTION: Assay
6. Incubate the mixture at room temperature for 20 minutes, preferably in the dark. 
SECTION: Assay
7. Rinse the cells with MilliQ water to remove excess dye. First, centrifuge the stained cells at 7000 RPM for 2 minutes. Remove about 900 µl dye with a 1 milliliter pipette (do not disturb the cell pellet). Since the dye is blue, the cell pellet cannot be seen during the first wash. Add 1 milliliter MilliQ water and centrifuge the cells. The cell pellet should be visible now as the leftover dye is no more concentrated. Repeat the above steps 2 to 3 times (remove 900 to 950 µl dye from the supernatant using a pipette, add MilliQ water, and centrifuge). Leave the cell pellet (after final wash) in a small volume (10 to 20 µl). Take a small amount of cell pellet (ca. 5 µl) and mount it on a clean glass slide. Put a coverslip, press gently so that cells are dispersed and observe the cells using a microscope. I use an oil immersion lens (100 X; Axioskop 40, Carl Zeiss, Germany). Count blue (dead) cells and estimate the dead cells (%) from the total cells.
TITLE: Calculating Colony-forming Units
AUTHORS: Jiri Hulcr, You Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to estimate the number of colony-forming units.</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: GFP pull down assay
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes GFP pull down assay.

[STEPS]
SECTION: GFP pull down assay
1. Mix GFP-tagged TBK1 with 20 µL of equilibrated GFP-Trap agarose beads (Chromotek) at a final  concentration of 1 micromolar (µM). Make sure to wash beads 2x in dH2O before washing with bead assay buffer to equilibrate the beads  adding the protein to the beads.
SECTION: GFP pull down assay
2. To this end, wash 20 µL of beads twice with dH2O and equilibrate with bead assay buffer. 
SECTION: GFP pull down assay
3. Resuspend the beads in 40 µL bead assay buffer, to this add GFP-TBK1 at a final concentration of 5 micromolar (µM).
SECTION: GFP pull down assay
4. Incubate the beads with GFP-TBK1 for 60 min at 4 °C at a horizontal tube roller.
SECTION: GFP pull down assay
5. Wash the beads three times to remove unbound GFP-tagged bait protein. 
SECTION: GFP pull down assay
6. Prepare protein master mixes with prey protein in bead assay buffer at the following concentrations: 
mCherry-OPTN (1 µM),
mCherry-NDP52 (1 µM), 
GST-NAP1 (1-10  µM). 
SECTION: GFP pull down assay
7. Add the protein master mixes to the beads and incubate for 60 min at 4 °C at a  horizontal tube roller.
SECTION: GFP pull down assay
8. Wash the beads three times to remove unbound proteins, remove any supernatant from the beads and resuspend the beads in 60 µL of 1x Protein Loading dye, and heat-inactivate at 95 °C for 5 min.
SECTION: GFP pull down assay
9. Analyze the samples by SDS-PAGE and Coomassie staining as described above.
TITLE: Processing wastewater samples for bacterial &amp; viral targets enrichment 
AUTHORS: Victor Mabasa

[DESCRIPTION]
This wastewater sample processing protocol by the Wastewater Genomics Syndicate at the National Institute of Communicable Diseases (NICD) is designed to enrich bacteria and viruses from the settled solids and supernatant components of wastewater, as different pathogens partition differently within this matrix. The end products are waterless solids and clarified wastewater supernatants, ready for downstream applications such as ultrafiltration, bead enrichment and nucleic acid extraction.   

[STEPS]
SECTION: Sample Collection &amp; Handling
1. Using correct personal protective equipment (PPE), collect a 1,000 ml grab wastewater sample from each location in a sterile bottle and transfer them to the laboratory in a cold chain (keep in a lab fridge at 4°C). it is important to proceed to the next step within the next 23 hours after sample collection to prevent major degradation of microorganisms. 
SECTION: Preparing for centrifugation
2. All work should be performed in a Class II biological safety cabinet while wearing appropriate PPE. Begin by cleaning your workspace with a 1% Virkon solution, followed by a 70% ethanol solution. Double-line your waste bucket with biohazard waste plastic, and ensure all your filter tips are refilled. Finally, label 500 mL conical centrifuge bottles, one for each wastewater sample.
SECTION: Centrifugation
3. Transfer 500 mL of the wastewater samples to the corresponding conical tubes

SECTION: Centrifugation
4. Centrifuge at 4 650 × g (deceleration speed set at 5), at 4oC for 10 minutes.
SECTION: Centrifugation
5. Carefully transfer the supernatant from the 500 mL conical bottle to a clean and marked 1,000 mL bottle without disturbing the settled solids.  
SECTION: Centrifugation
6. Add the remaining 500 mL of the original wastewater sample to the conical bottle with settled solids and repeat step 4. 
SECTION: Centrifugation
7. Carefully transfer the supernatants to 1,000 mL bottles in step 5, without disturbing the pellet. Keep at 4oC for different downstream applications (check our page for different nucleic acid extraction protocols from clarified wastewater) 
SECTION: Transfer settled solids
8. Use clean, plain wooden sticks to pick out settled solids and pack them into marked 2 mL cryo-tubes, aiming for approximately 250 mg per tube. 
SECTION: Transfer settled solids
9. Store the settled solids in the –80°C freezer until needed for downstream applications. (check our page for different nucleic acid extraction protocols from settled solids)