eamag/protocols-with-steps huggingface dataset:
TITLE: Environmental DNA Metabarcoding- Full Pipeline Collection
AUTHORS: Colleen Kellogg, Matt Lemay, rute.carvalho Carvalho, Andreas Novotny

[DESCRIPTION]
This is a collection of protocols used to analyze environmental DNA (eDNA) of seawater samples.
As part of the Hakai Institute Ocean Observing Program, biomolecular samples have been collected weekly from 0 m to near bottom (260 m), to genetically characterize plankton communities in the Northern Salish Sea since 2015. These protocols are developed to work across all domains of life, from viruses to prokaryotes to eukaryotes, allowing for both amplicon sequencing and shotgun sequencing.

This pipeline includes the following steps:
1. Seawater filtration: Our standard protocol for filtering seawater on sterivex filters. Alternatives may be used.

2. DNA extraction from sterivex filters: Here we offer two alternatives:
The robust Phenol Chloroform extraction protocol is used in our long-term monitoring program.
A kit alternative requires less handling of toxic chemicals, and are used for stand-alone projects.

3. DNA metabarcoding, library prep, and sequencing: With the options of analyzing different marker genes:
18S rRNA nuclear gene: Diversity of Eukaryotes. (Balzano et al 2015)
COI mitochondrial gene: Diversity of marine invertebrates. (Leray et al 2013)
12S rRNA mitochondrial gene: Diversity of fish. (Miya et al 2015)

4. Bioinformatic analyses
Examples bioinformatic analysis for the different genes can be found on our GitHub page: https://github.com/hakaigenomics

More information to be found under each protocol:


[STEPS]
TITLE: Slant board seed gemination and transplanting for hydroponic research
AUTHORS: noah.langenfeld  , Bruce  Bugbee

[DESCRIPTION]
Uniform seed germination is essential for hydroponic research. Conventional seed germination uses substrates such as peat moss or mineral wool. These substrates are difficult to remove from the roots and can lead to unwanted particulates in the nutrient solution following transplanting. Removal of substrates prior to transplanting damages fine root hairs and stunts growth. Germinating seeds on a slant board eliminates these problems. Root growth on a slant board is uniform, unobstructed, and reduces the time to transplanting. The long and straight roots also maximize the exposure of the taproot to the nutrient solution. The slant boards are made from vertically oriented acrylic sheets with germination paper on top. Seeds are held in place by thin cellulose paper. Seedlings can be transplanted with long, clean roots free from damage to root hairs. Here, we provide an overview of slant board construction and use for rapid seed germination and transplanting into hydroponic systems.

[STEPS]
SECTION: Seeding
1. Obtain a clean acrylic slant board and lay flat on a hard, clean surface.
SECTION: Seeding
2. Cut a piece of germination paper 10 cm in height and the same length as the slant board.
SECTION: Seeding
3. Place germination paper on top of slant board.
SECTION: Seeding
4. Place seeds on top of germination paper (about 9 cm from the bottom of the board and 1 cm from the top of the germination paper) in a line parallel to the top and bottom of the slant board. Seeds should be about 5 mm apart.

SECTION: Seeding
5. Place a single layer of cellulose paper (Kimwipe) over the seeds. 
SECTION: Seeding
6. Gently wet the entire germination board and Kimwipes with tap water to lock seeds and paper in place on the board. A squirt bottle works well for this process. Do not use nutrient solution.
SECTION: Seeding
7. Transfer the slant board into the exterior container. The angle of the board should be between 70° and 80°.
SECTION: Monitoring and transplanting
9. After at least one cotyledon leaf has appeared, gently peel back the Kimwipe to expose the leaves. The Kimwipe should remain covering the roots.
SECTION: Seeding
8. Fill the exterior container with tap water until the water level is about 2 cm above the bottom of the germination paper.
SECTION: Monitoring and transplanting
10. After the roots have grown long enough to reach the solution, remove the seedlings from the paper. Fingers or a tweezers can be used to remove the seedlings from the paper. Avoid excessive pressure to prevent root breakage.
SECTION: Monitoring and transplanting
11. Place the seedling inside a neoprene collar. Ensure the roots are not pinched in the collar. The cotyledons can be used to support the seedling and stop it from falling into the solution.
SECTION: Monitoring and transplanting
12. Place the neoprene collar with the seedling into the main culture tank. Ensure to roots are able to reach the nutrient solution to avoid desiccation.
TITLE: Deposition of matrix using an M5 TM sprayer for high resolution MALDI analysis
AUTHORS: Angela   Kruse, Martin  Dufresne, Jamie  Allen, Danielle  Gutierrez, Jeff  Spraggins

[DESCRIPTION]
This protocol describes the use of an HTX M5 TM sprayer to deposit small molecule matrices onto tissue sections for high resolution MALDI imaging mass spectrometry (IMS). Spraying conditions will vary depending on matrix, and this protocol focuses on matrices for lipid IMS such as DMACA and DHB. 

[STEPS]
SECTION: Autofluorescence Scan
1. Remove slides from freezer and place in desiccator for 30 min 

SECTION: Autofluorescence Scan
2. Collect autofluorescence scan using a Zeiss AxioScan Z1 (https://www.protocols.io/edit/autofluorescence-microscopy-data-acquisition-b39kqr4w)
SECTION: TM Sprayer Setup
3. Set nitrogen flow to 8 psi. turn on TM sprayer. 
SECTION: TM Sprayer Setup
4. Open HTX software
SECTION: TM Sprayer Setup
5. Set nozzle temperature to 60C, and heated stage temperature (if applicable) to 45C.
SECTION: TM Sprayer Setup
6. Clean sample loop with 10mL of methanol with sprayer set to "load". Switch to "spray" and allow to spray for 2 minutes.
SECTION: TM Sprayer Setup
7. Make matrix solution of DMACA or DHB at a concentration of 12mg/mL in a solution of 60% THF and 40% acetone containing 5uL/mL DMF. Sonicate solution for 10 minutes.
SECTION: TM Sprayer Setup
8. Switch sprayer to "load" and load sample loop with 2 mL of matrix solution using a syringe.
SECTION: TM Sprayer Setup
9. Switch sprayer to "spray" and allow to spray for ~30 seconds. Check that matrix is spraying by placing a glass slide under the nozzle. 
SECTION: TM Sprayer Setup
10. Tape slide onto heated stage, and adjust the spray area in the HTX software. 
SECTION: TM Sprayer Setup
11. Set the method in the HTX software:
1350 mm/min nozzle velocity
1.5 mm track spacing
0.05 mL/min flow rate
CC pattern
4 passes
40 mm nozzle height
No drying time
SECTION: TM Sprayer Setup
12. Ensure that the method is highlighted. Press start under the Cycle tab. Click Continue. Wait for purge delay and confirm that the nozzle is moving
SECTION: TM Sprayer Setup
13. When matrix deposition is finished, remove the slides and store them in a slide box. Press "valve load confirm" in the software.
SECTION: Cleanup
14. Set TM sprayer nozzle and stage temperatures to 30C
SECTION: Cleanup
15. With sprayer set to "load", load loop with 10 mL each of acetonitrile followed by methanol. Set sprayer to "spray" and allow to flow for ~5 minutes. 
SECTION: Cleanup
16. Spray nozzle tip with methanol, taking care not to touch the tip. 
SECTION: Cleanup
17. Clean the stage with methanol. Do not spray solvents directly onto the stage. 
SECTION: Cleanup
18. Replace the bench liner in front of the instruments
SECTION: Cleanup
19. Dispose of syringes in biohazard container
SECTION: Cleanup
20. Shut down HTX software and close the computer. Turn off the TM sprayer.
SECTION: Cleanup
21. Shut off the nitrogen flow.
TITLE: Systemic AAV vectors for widespread and targeted gene delivery in rodents
AUTHORS: Rosemary C. Challis, Sripriya Ravindra Kumar, Ken Y. Chan, Collin Challis, Keith Beadle, Min J. Jang, Hyun Min Kim, Pradeep S. Rajendran, John D. Tompkins, Kalyanam Shivkumar, Benjamin E. Deverman, Viviana Gradinaru

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We recently developed adeno-associated virus (AAV) capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. However, a detailed protocol for generating and systemically delivering novel AAV variants was not previously available. In this protocol, we describe how to produce and intravenously administer AAVs to adult mice to specifically label and/or genetically manipulate cells in the nervous system and organs, including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans 8 d, excluding the time required to assess gene expression, and can be readily adopted by researchers with basic molecular biology, cell culture, and animal work experience. We provide guidelines for experimental design and choice of the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.</div></div>

[STEPS]
?.
TITLE: Background and Rationale (Part 1 of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a collection of protocols for: "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults ≥ 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: Lysosomal Membrane Permeability (LMP) assay
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details the requirements of the Lysosomal Membrane Permeability (LMP) assay.

[STEPS]
SECTION: Cell Culture/Treatments:
1. 0.5x10^4/ml of cells plated on coverslips.
SECTION: Imaging:
6. Leica Stellaries 8 confocal laser scanning microscope.
SECTION: Cell Culture/Treatments:
2. PFF-488 treatment.
SECTION: Cell Culture/Treatments:
3. PBS/trypsin (0.01%) wash.
SECTION: Cell Culture/Treatments:
4. Fix cells using 4% PFA.
SECTION: Cell Culture/Treatments:
5. Immunostaining using Galectin 3 primary antibody.
SECTION: Imaging:
7. Image analysis: 

Image J. quantification of the % area of the cell covered by Galectin 3 immunoreactivity.
TITLE: Shipping Embryos in a Portable Incubator
AUTHORS: Eliab Estrada Cortés, Peter Hansen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol to describe how to ship non-frozen embryos</div></div>

[STEPS]
?. [ ]
Test the shipping incubator and make sure that it can remain powered for at least 24 hours while operating with the battery. We currently use TO 42 from WTA https://www.wtavet.com.br/?lang=en but others work well also. The temperature is set to 38.5°C
?. [ ]
Start charging shipping incubator 1 day before shipping so that it is fully charged at the time tubes containing embryos are added to the incubator.
?. [ ]
Make shipping label. We routinely use Fedex service with Priority Overnight Service. Fedex states that these packages arrive by 10:30 AM but it is more like noon for locations not close to a Fedex facility.  First Overnight Service is supposed to arrive by 8:00 am but arrival before 10:30 AM is more like it when the location is remote.  Note First Overnight is expensive!  We deliver the package to Fedex for shipping by 4:30 PM
?. [ ]
Two methods have been used successfully.Use 1 ml Corning cryogenic vials with orange caps filled with HEPES-TALP supplemented with 10% (v/v) fetal bovine serum (R&D Systems) and 50 µM dithithreitol. We have successfully shipped up to 15 embryos using this method.  Add up to ∼50 embryos per tube in polypropylene-stoppered 10 × 63 mm polystyrene tubes containing 1 ml SOF-BE2 supplemented with 1 mM HEPES, pH 7.5, and 50 μM dithiothreitol. Tubes are equilibrated overnight in a humidified incubator at 38.5°C and 5% O2, 5% CO2, and 90% N2. After addition of embryos to the medium, ∼1.5 ml mineral oil was layered over the medium and tubes were placed in the incubator.
?. [ ]
We have had plastic shipping containers custom-made for us by Benson Box (https://bensonbox.com) to protect the incubator during shipment. These are lined with Styrofoam to cushion the incubator and have lots of labeling to recommend caution with handling. The one currently used for the WTA incubator was designed for a large incubator and so additional padding is added to the inside of the container. Images are shown on the next page.Benson Boxes are very durable. 
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Here we describe the standardised protocol used by the <a href="http://www.caboscience.org" target="_blank" rel="noopener noreferrer">Canadian Airborne Biodiversity Observatory</a> (CABO) to measure leaf water content and specific leaf area, using the <a href="http://regent.qc.ca/assets/winfolia_about.html" target="_blank" rel="noopener noreferrer">WinFOLIA</a>™ software (<a href="http://regent.qc.ca/index.html" target="_blank" rel="noopener noreferrer">Régent Instruments</a>). These leaf area and water measurements are done on a subset of leaves from the same bulk leaf sample used to measure <a href="https://dx.doi.org/10.17504/protocols.io.pzbdp2n" target="_blank" rel="noopener noreferrer">leaf spectral reflectance and transmittance</a>. Briefly, after removing their petioles, fresh leaves are weighed, rehydrated for 6 h, scanned for total leaf area and weighed again; they are then oven-dried at 65 ºC for 72 h, and weighed one last time. This allows us to measure leaf dry matter content and its complement, leaf water content, as well as leaf relative water content. Leaf area measurements are used to estimate specific leaf area, a key functional trait central to the leaf economics spectrum. Specific leaf area allows us to estimate equivalent water thickness and to convert concentrations of foliar biochemical constitutents from a leaf mass to a leaf area basis.</p>

[GUIDELINES]
<p><strong>Equipment</strong></p>
<ul>
<li>Sartorius Secura 213-1S 1 mg (3 decimal places) balance or other similar balance</li>
<li>Sartorius Secura 1102-1S 0.01 g (2 decimal places) balance or other similar balance</li>
<li>Canon LiDE 220 portable scanner or other suitable flatbed scanner</li>
<li>WinFOLIA™ leaf area software (Régent Instruments Inc.)</li>
<li>Forced air drying oven</li>
<li>Dessicator</li>
<li><a href="https://www.edmundoptics.com/test-targets/color-gray-level-test-targets/rez-checker-matte/" target="_blank" rel="noopener noreferrer">RezChecker</a> color/resolution target (optional)</li>
</ul>
<p><strong>Consumables</strong></p>
<ul>
<li>Paper towels</li>
<li>Paper envelopes and/or bags</li>
<li>Stapler and staples (for paper bags)</li>
<li>Sealed plastic bags</li>
<li>Weighing trays</li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Quick Protocol for Monarch® Plasmid Miniprep Kit (NEB #T1010)
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is the quick version of the Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010). For the full protocol, please click </span><a href="https://www.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">here</span></a><span>.</span></div></div>

[STEPS]
?. Pellet 1–5 ml (not to exceed 15 OD units) bacterial culture by centrifugation for 30 seconds. Discard supernatant.
1.5 ml of culture is sufficient for most applications. Ensure cultures are not overgrown (12-16 hours is ideal).
?. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1).
Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
?. Add 200 μl Plasmid Lysis Buffer (B2), gently invert tube 5–6 times, and incubate at room temperature for 1 minute. Do not vortex.
[incubation]
Color should change to dark pink, and solution will become transparent and viscous. Do not vortex.
?. Add 400 μl of Plasmid Neutralization Buffer (B3), gently invert tube until neutralized, and incubate at room temperature for 1 minute. Do not vortex.
[incubation]
Sample is neutralized when color is uniformly yellow and precipitate forms. Do not vortex.
?. Centrifuge lysate for 2–5 minutes.
[centrifugation]
For culture volumes >1 ml, we recommend a 5 minute spin to ensure efficient RNA removal by RNase A. Pellet should be compact; spin longer if needed.
?. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
[centrifugation]
?. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Centrifuge for 1 minute.
Discarding the flow-through is optional.
?. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
[centrifugation]
?. Transfer column to a clean 1.5 ml microfuge tube.
Use care to ensure that the tip of the column does not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute.
?. Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
[wait]
[spin]
Nuclease-free water (pH 7–8.5) can also be used to elute the DNA. Yield may slightly increase if a larger volume of DNA Elution Buffer is used, but the DNA will be less concentrated. For larger size DNA, (≥ 10 kb), heating the elution buffer to 50°C prior to use can improve yield.
TITLE: Stereotactic Injections in Mouse and Rat
AUTHORS: Joris Van Asselberghs, Veerle Baekelandt

[DESCRIPTION]
This protocol outlines procedures for Stereotactic Injections in Mouse and Rat.

[STEPS]
SECTION: Procedure
1. Remove hair and put 2 % on top of the head and into the ears.
SECTION: Procedure
2. Put Vidisic on the eyes.
SECTION: Procedure
3. Put a chip under the skin to mark the animal.
SECTION: Procedure
4. Fix the animal into the stereotactic apparatus (use the mouse adaptor for mouse and only the ear clamps for rat). Put a tissue over the animal to keep it warm during surgery.
SECTION: Procedure
5. Make sure that the left and right side of the skull is positioned as straight as possible (ears).
SECTION: Procedure
6. Use 1 % to clean the top of the head and make an incision with a scalpel.
SECTION: Procedure
7. Clean the skull with a spatula and saline and let dry until bregma and lambda are clearly visible.
SECTION: Procedure
8. Check volume and injection speed of the pump, rinse Hamilton syringe with RBS, ETOH and PBS. (Coat the syringe by taking a full syringe of vector and discard in eppendorf). Put vector in the syringe (± 1.5 µl more than you want to inject) and make sure there are no air bubbles.
SECTION: Procedure
9. Make sure bregma and lambda have the same height, correct the position of the head if there’s a difference of more than 0.02cm.
SECTION: Procedure
10. Put the needle at the right position using bregma as a reference (find the right coordinates using the stereotactic atlas of mouse or rat).
SECTION: Procedure
11. Drill a small hole into the skull at this position until the dura mater is visible.
SECTION: Procedure
12. Make a small hole into the dura mater using a thin needle.
SECTION: Procedure
13. Put the Hamilton needle at the right position (go down slowly to prevent tissue damage) and wait for 1 min.
SECTION: Procedure
14. Inject vector at max. 0.25 µl/min.
SECTION: Procedure
15. Wait 5 min after injection in order to let the vector diffuse into the brain.

SECTION: Procedure
16. Remove the needle slowly.
SECTION: Procedure
17. Close the skin and disinfect with joodalcohol.
SECTION: Procedure
18. Rinse Hamilton syringe with RBS, ETOH, PBS and AD.
TITLE: Chitin binding assay using chitin magnetic beads (NEB)
AUTHORS: Robert Hooftman

[STEPS]
?. Make the following buffer:1 X CBD Column Binding Buffer  ABC1Tris-HCl&nbsp;20 mM&nbsp;Adjust pH to 8&nbsp;using HCl&nbsp;(2,42 g/L)&nbsp;2NaCl&nbsp;500 mM&nbsp;&nbsp;(29,22 g/L)&nbsp;3EDTA&nbsp;1 mM&nbsp;&nbsp;(0,292 g/L)&nbsp;4Tween-20&nbsp;0.05% v/v&nbsp;500 ul/ 1 L&nbsp; Gently mix the beads solution and resuspend thoroughly.
ABC1Tris-HCl&nbsp;20 mM&nbsp;Adjust pH to 8&nbsp;using HCl&nbsp;(2,42 g/L)&nbsp;2NaCl&nbsp;500 mM&nbsp;&nbsp;(29,22 g/L)&nbsp;3EDTA&nbsp;1 mM&nbsp;&nbsp;(0,292 g/L)&nbsp;4Tween-20&nbsp;0.05% v/v&nbsp;500 ul/ 1 L&nbsp;
?. Aliquot 50 ul of beads solution into a 2 ml microfuge tube
?. Apply a magnet for 30 seconds to pull the beads to one side. Remove the excess liquid
?. Wash the beads 3 times with 500 ul of 1x CBD Column Binding Buffer. Apply magnet and remove the liquid.
?. Load 200-500 ul of cell supernatant with the beads.Note: when making dilutions of the supernatant, dilute in CBD Column Binding Buffer.
?. Mix thoroughly and incubate for 1 hour at 4°C at constant agitation.
?. Apply magnet for 30 seconds and remove supernatant with a pipette. Save the supernatant.
?. Wash beads 3 times with 500 ul of 1x CBD Column Binding Buffer. Apply magnet for 30 seconds and pipette off liquid. Save the wash fractions.
?. To remove proteins from the beads, boil beads for 5 minutes in 50 ul SDS-Page sample buffer at 95 °C
?. Run fractions on a protein gel
TITLE: Tutorial for MATLAB functions for chicken behaviors
AUTHORS: Alireza Abdoli, Amy Murillo, Alec Gerry, Eamonn Keogh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This tutorial describes the library of key functions, developed in MATLAB, to identify chicken behaviors: pecking, preening, and dustbathing. The classification algorithm was developed based on combination of shapes and features as discussed in Abdoli et al. (2020) and was used for data analyses in Murillo et al. (2020).</div></div>

[STEPS]