eamag/protocols-with-steps huggingface dataset:
TITLE:         ELISA for quantification of human C5 in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. An anti-human C5 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human C5 present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-C5 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-C5 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 20 min.
?. A colored product is formed in proportion to the quantity of C5 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human C5 standard dilutions and the human C5 sample concentration is determined.
TITLE: JAX-Sen: Collection and shipment of specimen for Bulk RNA-sequencing (Bulk RNA-seq)
AUTHORS: Laura Robinson, Susan Sheehan, Gaven Garland, Ron Korstanje

[DESCRIPTION]
These samples are part of the JAX-Sen project in the SenNet Consortium. Here we provide details on specimen collection and shipment to the Robson laboratory at The Jackson Laboratory for Genomic Medicine (JAX-GM) in Farmington, CT for its processing for Bulk RNA-sequencing (Bulk RNA-seq).

[STEPS]
SECTION: Reagents and Materials:
1. 2mL Eppendorf tubes or 5 ml Eppendorf tubes
RNA Later solution
Wet Ice
Tweezers (clean, sterile)
SECTION: Quality Key Points:
2. The tissue specimen should be always kept at 4 degrees Celsius and RNase-free. 
It is crucial to not store the tissue specimen at RT to avoid any cell death, and tissue and/or RNA degradation. 
SECTION: Procedure:
3. Timeline: The daily shipping deadline at Jax BH is 12:00 noon. So, harvest the tissues before and closer to noon.
SECTION: Procedure:
4. Collection/Harvest:
SECTION: Procedure:
4.1. Animal was euthanized via cervical dislocation. 
SECTION: Procedure:
4.2. Animal was pinned to a necropsy tray which remained on wet ice throughout the harvest. 
SECTION: Procedure:
4.3. Animal was perfused with 20ml cold PBS.
SECTION: Procedure:
4.4. Heart, pancreas, and kidney was collected and the specimen was transferred into tubes containing ice-cold RNA later (the volume will depend on the size of the specimen, but should cover abundantly the tissue and fill up the tube)
SECTION: Procedure:
4.5. Individually parafilm the Eppendorf tube lids to their tubes before shipping.
SECTION: Procedure:
4.6. Keep at 4 degrees Celsius thereafter, until shipping.
SECTION: Procedure:
5. Shipping:
SECTION: Procedure:
5.1. Place sample tubes in a plastic box (cardboard boxes insulate the samples from the cold ice) or in double Ziplock bags after checking that they are completely sealed.
SECTION: Procedure:
5.2. Ship the sample box on wet ice.
SECTION: Procedure:
5.3. Ship O/N on ice/ice pack, to:
The Jackson Laboratory for Genomic Medicine,
Farmington, CT, 06032
TITLE: DNA quantification using the Quantus fluorometer 
AUTHORS: Josh Quick

[STEPS]
?. Set up the required number of  tubes for the number of DNA samples to be quantified.
0.5 ml
Use only thin-wall, clear, 0.5mL PCR tubes such as Axygen #PCR-05-C
?. Label the tubes on the lids, avoid marking the sides of the tube as this could interfere with the sample reading.
?. Add  ONE dsDNA dye solution to each tube.
199 µl
?. Add  of each user sample to the appropriate tube.
1 µl
Use a P2 pipette for highest accuracy.
?. Mix each sample vigorously by vortexing for  and pulse centrifuge to collect the liquid.
?. Allow all tubes to incubate at room temperature for before proceeding.
?. On the Home screen of the Quantus Fluorometer, select `Protocol`, then select `ONE DNA` as the assay type.
If you have already performed a calibration for the selected assay you can continue, there is no need to perform repeat calibrations when using ONE DNA pre diluted dye solution. If you want to use the previous calibration, skip to step 11. Otherwise, continue with step 9.
?. Set up two  tubes for the calibration and label them 'Blank' and 'Standard'
0.5 ml
?. Mix the Lambda DNA standard 400 ng/µL standard by pipetting then add  to one of the standard tube.
1 µl
?. Selection 'Calibrate' then 'ONE DNA' then place the blank sample in the reader then select 'Read Blank'. Now place the standard in the reader and select 'Read Std'.
?. On the home screen navigate to 'Sample Volume' and set it to then 'Units' and set it to ng/µL.
1 µl
?. Load the first sample into the reader and close the lid. The sample concentration is automatically read when you close the lid.
?. Repeat step 16 until all samples have been read.
?. The value displayed on the screen is the dsDNA concentration in ng/µL, carefully record all results in a spreadsheet or laboratory notebook.
?. Remove Lambda DNA 400 ng/µL standard from the freezer and leave on ice to thaw. Remove ONE dsDNA dye solution from the fridge and allow to come to room temperature.
?. Add  ONE dsDNA Dye solution to each tube.
200 µl
?. Mix each sample vigorously by vortexing for  and pulse centrifuge to collect the liquid.
?. Allow both tubes to incubate at room temperature for  before proceeding.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Overview:</p>
<p>1. Filter DNA reads by quality and host (e.g. for mouse gut bacteria we would filter low-quality reads AND reads that belong to mice)</p>
<p>2. Align DNA reads (fastq, fasta) to <a href="https://www.patricbrc.org/" target="_blank">Patric</a> database (bacteria genomes) with <a href="https://github.com/r78v10a07/taxoner" target="_blank">taxoner</a> (a bowtie2-based linux program) &gt;&gt; Get bacterial genomes</p>
<p>3. Filter out low-quality alignments &gt;&gt; Bacterial genomes</p>
<p>4. Filter RNA reads by quality</p>
<p>5. Align RNA reads to bacterial genomes from step 3 &gt;&gt; RNA counts of genes</p>
<p>6. Get pathway information &gt;&gt; RNA counts per KEGG pathway</p>
<p> </p>
<p>If you have treatment groups, we will be able to see changes in bacterial abundance as well as changes to RNA counts</p>

[BEFORE_START]
<p>Unless you have a small set of reads and/or genomes, it is recommended that the pipeline be run on university supercomputers.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The aim of this study was to analyze the trend in mortality from uterine neoplasms and their risk factors, specified by the International Classification of Diseases - 10th Edition (ICD-10): C53, C54 and C55. This is an ecological study of a cross-sectional cohort and time series of deaths from uterine cancer that were recorded on the death certificates of 147 women residing in Maringá-PR from 2004 to 2014. Deaths attributed to malignant neoplasms of the cervix, unspecified (C53.9) were the most common among these women. There was an increasing trend in mortality rates from cancer of the uterus. The behavior of age groups showed an increase in women over 65 years of age. Overall, 71.5% of the women had less than 8 years of education, and 60.5% had no partner. It was also found that the health unit in Vardelina had the highest number of deaths, corresponding to 0.73% of the population. The percentage of the total deaths in the city was 0.15%. These data indicate that women in lower social strata have less access to health services and less information regarding primary prevention and the effective treatment of uterine cancer.</p>

[STEPS]
TITLE: ISP Fish Biodiversity Survey
AUTHORS: Stephanie Gay, Kylie Ren, Charlotte Wiggins

[DESCRIPTION]
This survey addresses the marine biodiversity present off the coast of the Onetahi motu in Tetiaroa, French Polynesia. Researchers conducted transects observing present fish species and their interactions in order to analyze the structure of the ecosystem's food web and overarching species richness. Biodiversity analysis is an integral contribution to the knowledge and subsequent preservation of marine environments, and research in small-scale ecosystems like Tetiaroa is representative of the larger environmental conservation movement.

[GUIDELINES]
- Be safe! Wear sunscreen, watch out for sharp objects when swimming, and have fun :D

[STEPS]
SECTION: Belt Transect Survey
2. Split into teams of 2 (with additional team member(s) to assist with securing the transect tape)
SECTION: Species Identification
6. Export photos from camera to laptop
SECTION: Species Interaction
9. Photograph distinct interactions between individuals of different species
SECTION: Survey Site Selection
1. From north shore of the Onetahi Motu, aligned with ferry departure port, swim outwards towards the reef crest until reef substrate becomes primarily coral (as opposed to sand) 
SECTION: Belt Transect Survey
3. Mark out a 30m transect section using transect tape
Secure one end of the tape using a rock/coral or team member to secure the tape
Assign one team member to swim out with the other end of the tape to mark the full 30m length - head in a fixed direction either with use of compass or by heading toward a fixed landmark visible at horizon level
SECTION: Species Identification
5. Photograph individuals of each unique species of fish sighted
SECTION: Belt Transect Survey
4. For member of team performing the swimming survey task
Swim forward at a slow constant speed along the tape
Estimate a transect width of 2m on either side of the tape
Within the transect, use camera to document what is required (refer to step 5 and 6)
Avoid splashing and move slowly to avoid scaring fish away
Pause and wait if necessary along transect to allow fish to acclimatize to your presence
SECTION: Species Identification
7. Identify fish species 
Compare each photo of fish obtained with the fish in guide book or https://www.tetiaroasociety.org/island/fish
SECTION: Species Identification
8. Record the Genus and Species name for each species of fish identified into a spreadsheet
SECTION: Species Interaction
11. Identify the species involved, and record description of these interactions in a spreadsheet marking them as trophic or non-trophic
SECTION: Species Interaction
12. Analyze relationships between species, record connections and hypothesized behavior behind interactions
SECTION: Species Interaction
10. Export photos from camera to laptop
SECTION: Species Function
13. Photograph individuals performing functions for each unique species of fish sighted
SECTION: Species Function
14. Identify the species involved, and record description of the function performed
TITLE: HTTM : gDNA extraction
AUTHORS: Antoine Champie

[DESCRIPTION]
Part of the HTTM protocol dedicated to the extraction of gDNA from transposon mutated cell pellets. 

[STEPS]
SECTION: DNA extraction
2. Add 600 µLof lysis solution to each well of the deep-well plate and resuspend the pellet. 

SECTION: DNA extraction
1. Prepare the lysis solution by adding 165 µL of proteinase K to 66 mL of homemade lysis buffer and mix well. 

SECTION: DNA extraction
3. Cover with an adhesive aluminum foil and incubate at 55 °C for 60 min. 

SECTION: DNA extraction
4. While still warm, add 260 µLof ethanol 100%, without overmixing.

 

SECTION: DNA extraction
5. Transfer immediately to a deep-well plate fitted with an array of silica columns. 
SECTION: DNA extraction
6. Centrifuge twice at 4000 x g, 10 min . 


SECTION: DNA extraction
7. Discard flowthrough and add 500 µL of wash solution.

SECTION: DNA extraction
8. Centrifuge at 3000 x g, 10 min 


SECTION: DNA extraction
9. Discard flowthrough.

SECTION: DNA extraction
10. Centrifuge at 3000 x g, 10 min  to eliminate traces of wash solution. 
SECTION: DNA extraction
11. Discard flowthrough.

SECTION: DNA extraction
12. Add a collector plate between the silica column array and the deep-well plate. 
SECTION: DNA extraction
13. Add 50 µLof low TE to the silica matrix in each well. 

SECTION: DNA extraction
14. Cover with an adhesive aluminum foil and incubate at55 °C for 15 min.

SECTION: DNA extraction
15. Centrifuge at 3000 x g, 10 min .

SECTION: Silica array regeneration (Optional)
16. Put the contaminated silica array on an empty deep-well plate.
Add 150 µL of 1N NaOH + 0.15%(v/v) Triton X-100 to each well. 

SECTION: Silica array regeneration (Optional)
17. Incubate at Room temperature for 5 min

SECTION: Silica array regeneration (Optional)
19. Add 200 µL of 1,5N HCl+ 0,15% (v/v) Triton X-100 to each well. 
SECTION: Silica array regeneration (Optional)
18. Centrifuge 3000 x g, 5 min 

SECTION: Silica array regeneration (Optional)
20. Incubate at Room temperature for 30 min 

SECTION: Silica array regeneration (Optional)
21. Centrifuge 3000 x g, 5 min 

SECTION: Silica array regeneration (Optional)
22. Add 150 µL of 1N NaOH + 0,15%(v/v) Triton X-100 to each well. 
SECTION: Silica array regeneration (Optional)
23. Incubate at Room temperature for 5 min

SECTION: Silica array regeneration (Optional)
24. Centrifuge 3000 x g, 5 min 
SECTION: Silica array regeneration (Optional)
24.1. Collect the flowthrough in a beaker. Neutralize pH if needed and dispose of the flow through. 
SECTION: Silica array regeneration (Optional)
25. Add 200 µLof ddH2O to each well. 

SECTION: Silica array regeneration (Optional)
26. Centrifuge 3000 x g, 5 min 
SECTION: Silica array regeneration (Optional)
27. Silica columns array are ready to be reused. 
SECTION: DNA extraction
8.1. Repeat steps 7 and 8. 
SECTION: Silica array regeneration (Optional)
26.1. Repeat steps 25 and 26.
TITLE: Croprological flotation techniques Tibor-Kassai, 1998
AUTHORS: Javier Antonio Benavides-Montaño, Javier Antonio Benavides

[STEPS]
?. Prepare a Sheather's Sucrose Solution. Density = 1.20-1.27. Preparation: dissolve over indirect heat (to avoid caramelization) approximately 500 g of granulated sugar (sucrose) in 1 liter of boiling water; add 6 ml of 37% formaldehyde or crystallized phenol to prevent microbial growth (mold).
?. Immerse 3-5 g of feces in water for 30 minutes; dissolve the stool balls. using a glass rod or an electric homogenizer until a semi-solid suspension is obtained.
?. Deposit the mixture in a centrifuge tube and centrifuge at 1500 rpm for 2 minutes, subsequently discarding the supernatant
?. Re-suspend the sediment completely by shaking
?. Fill the tube with one of the flotation solutions. mix the contents, covering the tube with your thumb and inverting it several times; do not shake, avoid the formation of air bubbles
?. Centrifuge again at 1500 rpm for 2 minutes
?. Place the drop where the eggs are on a slide, lightly pressing the glass rod or the handle on it; add a drop of water, place a coverslip and examine the entire preparation under the microscope using the 10X objective; using the 40X objective to appreciate in detail the morphology of the eggs
TITLE: Artificial seawater medium
AUTHORS: Shaaijer, l.kop, h.koch, Sluecker

[DESCRIPTION]
This artificial seawater medium was adapted from the Synthetic Crenarchaeota Medium described in Könneke et al. 2005 (10.1038/nature03911).

[STEPS]
SECTION: SCMU medium
2. Dissolve in 900 ml demineralized water:
 

SECTION: SCMU medium
3. Autoclave at 121°C for 20 min at 15 psi.
SECTION: SCMU medium
4. After cooling to room temperature add (in the flow cabinet) from sterile stocks:
 

SECTION: SCMU medium
5. Adjust the pH to 7.8 using sterile 1M NaHCO3 solution.
SECTION: Thauer&#x27;s vitamin solution
6. Dissolve in 1l demineralized water:
 

SECTION: Thauer&#x27;s vitamin solution
7. Adjust to pH 7.0 with KOH.
SECTION: Thauer&#x27;s vitamin solution
8. Filter sterilize (0.2 µm) and store at 4ºC.
SECTION: Trace Elements Solution I (TES I)
9. Dissolve 10 g NTA (Trisodium Nitrilotriacetate) in 500 ml demineralized water.
SECTION: Trace Elements Solution I (TES I)
13. Filter sterilize (0.2 µm) or autoclave at 121°C for 20 min at 15 psi. 
SECTION: Trace Elements Solution I (TES I)
14. Store in the dark (slightly light sensitive) at RT.
SECTION: Trace Elements Solution II (TES II)
17. Add the following ingredients and dissolve:
 

SECTION: Trace Elements Solution II (TES II)
19. Filter sterilize (0.2 µm) or autoclave at 121°C for 20 min at 15 psi.
SECTION: Trace Elements Solution II (TES II)
20. Store in the dark (slightly light sensitive) at RT.

1. Prepare the Thauer's vitamin solution and trace element solutions according to the recipes below. Aliquot for future use.
SECTION: Trace Elements Solution I (TES I)
10. Adjust to pH 8 using NaOH pellets.
SECTION: Trace Elements Solution I (TES I)
11. Add 5 g FeSO4 x 7 H2O and dissolve.


SECTION: Trace Elements Solution I (TES I)
12. Top up with demineralized water to a total volume of 1l.
SECTION: Trace Elements Solution II (TES II)
18. Top up with demineralized water to a total volume of 1l.
SECTION: Trace Elements Solution II (TES II)
16. Adjust to pH 8 using NaOH pellets.
SECTION: Trace Elements Solution II (TES II)
15. Dissolve 5.5 g NTA (Trisodium Nitrilotriacetate) in 500 ml demineralized water.
TITLE: M9 minimal medium
AUTHORS: Isaac Núñez, Tamara Matute

[DESCRIPTION]
M9 minimal medium is a classical bacterial growth broth (Miller, et al., 1972) characterized by its low autofluorescence. 
As it is minimal, it can be supplemented with defined carbon sources or amino acids acord the custom experimental setups.



[STEPS]
SECTION: Recipe
1. This is the recipe for 500 mL total medium.
It is assembled from sterile reagents in sterile conditions (in a laminar flow or flame, and using a sterille glass bottle). They have to be added in this order and mixed well after incopore each component.

250 mL Sterile Molecular grade water
100 mL 5X M9 Salt Mix
1 mL MgSO4:7H2O  (1M)
10 mL Glucose (20% w/v)  --> 0.4 w/v final concentration in the medium
50 µL CaCl2 (1M)
100 mL Casaminoacids (1%)
complete with ~39 mL Sterile Molecular grade water

In case of solid media preparation:
39 mL Sterile Molecular grade water
100 mL 5X M9 Salt Mix
1 mL MgSO4:7H2O (1M)
10 mL Glucose (20% w/v) --> 0.4 w/v final concentration in the medium
50 µL CaCl2 (1M)
100 mL Casaminoacids (1%)

melt Agar 3% in a microwave or hot bath and complete the media with 250 mL of it. 
It should be added quite hot to allow you mix it well and give you enough time to do the plates before it gets solid.

Antibiotic supplementation can be done after adding the Agar.

Plates could be stores at 4°C and darkness for a couple of days but sometimes they form precipitate.

*** You can replace the carbon source or aminoacids for any other(s) accord your experimental setup ***
SECTION: Reagents preparation
7. Agar 3% - 500 mL
Weight 15 g of Agar (molecular biology grade).
Complete to 500 mL with deionized water and dissolve completely.

Sterilize by autoclaving.

SECTION: Reagents preparation
6. Casaminoacids (1%) - 100 mL
Weight 1 g of Casaminoacids.
Complete to 100 mL with Molecular grade or deionized water and dissolve completely.

Sterilize by autoclaving.

SECTION: Reagents preparation
5. CaCl2 (1M) - 5 mL
Weight 0.555 g of CaCl2.
Complete to 5 mL with Molecular grade or deionized water and dissolve completely.

Sterilize by filtering with 0.22 µm syringe filter.
SECTION: Reagents preparation
4. Glucose (20% w/v) - 100 mL
Weight 20 g of Glucose.
Complete to 100 mL with Molecular grade or deionized water and dissolve completely.

Sterilize by filtering with 0.22 µm syringe filter.

SECTION: Reagents preparation
3. MgSO4:7H2O (1M) - 20 mL
Weight 4.930 g of MgSO4:7H2O
Complete to 20 mL with Molecular grade or deionized water and dissolve completely.

Sterilize by autoclaving

SECTION: Reagents preparation
2. 5X M9 Salts (600 mL)

Weight these amounts of each salt and add to a glass bottle:

38.4 g Na2HPO4:7H2O
9 g KH2PO4
1.5 g NaCl
3 g NH4Cl

Then, add 600 mL Molecular grade or deionized water and dissolve completely.

Sterilize by autoclaving
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a RNA extraction protocol adapted from Byron Crump's lab, which was developed from both Zhou et al. 1996 and Crump et al. 2003. It is intended to be used with 47 mm 0.22 um PES filters or a 0.22 uM Sterivex filter, both preserved with RNALater after sample collection.</p>

[STEPS]
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TITLE: Advancements and Critical Steps for Statistical Analyses in Blood Pressure Response to Resistance Training in Hypertensive Older Women:  A Methodological Approach
AUTHORS: Dahan Cunha Nascimento

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This document represents a supplemental appendix material for a better understanding of preprocessing steps of all statistical analysis used in the study </span><span style = "font-weight:bold;">"Advancements and Critical Steps for Statistical Analyses in Blood Pressure Response to Resistance Training in Hypertensive Older Women: A Methodological Approach".</span></div></div>

[STEPS]
TITLE: Animal housing and ethical concerns
AUTHORS: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino

[DESCRIPTION]
This protocol details animal housing and ethical concerns.

[STEPS]
SECTION: Animal housing and ethical concerns
1. Males C57BL/6J wild type (WT) and C57BL/6J LRRK2*G2019S 2AMjff/J transgenic (TG-G2019S) (stock 018785) mice purchased from Jackson Laboratory (Bar Harbor, ME, USA) at 8-12 weeks of age were housed at the animal facilities unit (C.A.Pi.R. Via Santa Sofia 97, Catania 13/2017-UT) and maintained on a 12:12-h light/dark cycle with ad libitum food and water. 
SECTION: Animal housing and ethical concerns
2. The animals were given 3 weeks to acclimate to the housing conditions prior to study commencement.
TITLE: Neuropathy Phentoyping Protocols - Dosages of Analgesics for Rats and Mice
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block"><span style = "font-weight:bold;">Phenotyping of Rodents for the Presence of Diabetic Neuropathy </span></div><div class = "text-block">In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bim84kcn.jpg" /></div></div>

[STEPS]