eamag/protocols-with-steps huggingface dataset:
TITLE: Data Handling and Recordkeeping (Part 8 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 Part 8 of "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: Electroporation
AUTHORS: Jiaxin Li

[STEPS]
?. Separate the bacterial liquid in shaking tube into 1.5 mL (2 tubes) per tube, centrifuge quickly for 90 s at 4℃ and 12000 rpm.
?. Add 1 mL of 300 mM sucrose solution to each tube of bacterial liquid, centrifuge for 2 min at 12000 rpm and 4℃, and pour it out.
?. Resuspend the competent cells (tube one) by adding 100 μl 300 mM sucrose solution, then transfer the liquid to tube two to resuspend the competent cells, and divide into 50 μl tubes (two tubes in total).
?. Add 1 μg plasmid into each tube, gently blow and mix.
?. Add 50 μl of suspension to the electroporation cuvettes.
?. [Electroporation]
Turn on electroporator and set to 3kv, 400 ohms and 25 µF
?. [Electroporation]
Take out the liquid culture medium of LB with 37℃ heat preservation for standby.
?. [Electroporation]
Transfer the DNA-cell mixture to the cold cuvette, tap on countertop, wipe water from exterior of cuvette and place in the electroporation module and press pulse.
?. [Electroporation]
Immediately add LB culture solution, mix by pipetting up and down once and transfer to a microcentrifuge tube, 5 ml culture tube.
?. Place in the shaker/incubator at 37°C incubator for 1 h-2 h
?. Incubate overnight at 37°C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for the cultivatio of Alternaria Solani.</p>
<p> </p>
<p>This protocol has been used for many years at the chair of Phytopathology at the Technical University of Munich.</p>
<p> </p>
<p>SNA Medium was first described by Nirenberg in 1981.</p>

[GUIDELINES]
<p>Work under sterile conditions when inoculating new plate.</p>

[STEPS]
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TITLE: DAB Detection of Biocytin Labeled Tissue
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the process for diaminobenzidine (DAB) detection of biocytin filled cells.  This protocol is optimized for use with brain slices cut at 350 µm thick, in which cells are first filled with biocytin (i.e., post-electrophysiological recording), fixed in 4% PFA/2.5% glutaraldehyde, and transferred to PBS until ready to stain.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides an efficient way to observe proteins in samples. </p>

[BEFORE_START]
<p>Prepare acrylamide gels a few hours before running the gels; acrylamide percentage depends on protein sizes. Prepare SDS-PAGE buffers beforehand as well. </p>

[STEPS]
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TITLE: Gene editing of YIPF3, YIPF4, CALCOCO1, FIP200, and ATG7 in HEK293 and HeLa cells V2
AUTHORS: Harper JW, Kelsey Hickey, sharan_swarup

[DESCRIPTION]
This protocol describes the creation of YIPF3, YIPF4, FIP200, CALCOCO1, and ATG7 knockout cell lines in HEK293 and HeLa cells using CRISPR-Cas9.

[STEPS]
SECTION: Cell line maintenance
1. Maintain HEK293 and/or HeLa cells in Dulbecco’ Modifies Eagles Medium (DMEM) with 10% fetal bovine serum and optional 1% penicillin-streptomycin. 
SECTION: Targeted knock-out specific genes including YIPF3, YIPF4, CALCOCO1, FIP200, or ATG7: targeting vector creation
2. For YIPF4 knock-out, the following sgRNA sequences were designed and ordered (5’  ATCTCGCGGCGACTCCCAAC 3' / 5’ CGGCCTATGCCCCCACTAAC 3' ), and cloned into a pX459 vector to create pX459-gRNA-YIPF4-KO.  

For CALCOCO1 knock-out, the following sgRNA sequences were designed and ordered (5’  AAGTTGACTCCACCACGGGA 3' / 5’ CTAAGCCGGGCACCATCCCG 3'), and cloned into a pX459 vector to create pX459-gRNA-CALCOCO1-KO. 

For ATG7 knock-out, the following sgRNA sequence was designed and ordered (5’ ATCCAAGGCACTACTAAAAG 3'), and cloned into a pX459 vector to create pX459-gRNA-ATG7-KO. 

For FIP200 knock-out, the following sgRNA sequence was designed and ordered (5’ ACTACGATTGACACTAAAGA 3'), and cloned into a pX459 vector to create pX459-gRNA-FIP200-KO. 

For YIPF3 knock-out, the following sgRNA sequences were designed and ordered (5’  CCATTTCGGGCGCCGCCCGC 3' / 5’ GGCGGCGCCCGAAATGGAGC 3'), and cloned into a pX459 vector to create pX459-gRNA-YIPF3-KO. 

Sequence validate by Sanger sequencing.
SECTION: CRISPR editing and confirmation
3. Transfect HEK293 and/or HeLa cells with the pX459-gRNA-YIPF4-KO, pX459-gRNA-CALCOCO1-KO, pX459-gRNA-ATG7-KO, gRNA-FIP200-KO, or gRNA-YIPF3-KO with Lipofectamine 3000, and select with 1.2 µg/mL of puromycin for 24-48 hours. Select monoclonal cells by limiting dilution or by cell sorting (SONY SH800S sorter) in 96 well plates. 
SECTION: CRISPR editing and confirmation
3.1. Individual clones were subjected to immunoblotting with anti-YIPF4 (Sino Biological 202844-T46), anti-ATG7 (Cell Signaling Technology, 8558S), anti-FIP200 (Cell Signaling Technology, 12436), anti-YIPF3 (Invitrogen PA566621), or anti-CALCOCO1 Rabbit mAb antibody (Abclonal A7987) and clones lacking the relevant protein were selected for further analysis by Sanger sequencing of the edited alleles.
TITLE:  Quick and simple removal of endotoxins from purified protein solutions with magnetic beads
AUTHORS: Gudrun Baersch

[DESCRIPTION]
Endotoxins (lipopolysaccharides) can affect the results of protein solutions in many ways, so it is important to eliminate them before in vitro and in vivo testing. There are several resin and chromatography- based solutions on the market. However, the use of these commercially available endotoxin elimination methods leads to significant losses of protein. 
E. coli produces endotoxins as it is utilized to express a variety of commercially accessible recombinant proteins. The presence of trace amounts of endotoxins has negative effects on proteinsolutions. Therefore, we have developed magnetic beads that eliminate such endotoxins when introduced into solutions. There is no loss of proteins in these applications. Magnetic beads offer a simple and fast way to remove these endotoxins. They are also cost-effective because the beads used can be regenerated and reused multiple times.

[BEFORE_START]
-Read the protocol before start.
-Check the contents of the kit.
-The product (magnetic beads) is sterile. 
-If the user want to have a sterile product, user should work under laminar flow. 

[STEPS]
SECTION: Working protocol
1. Performing of the isolation:
The user should use 10µl of magnetic beads for 250µl solution, from which the endotoxins are to be removed. If the user has 1 ml of solution, 40µl of magnetic beads should be used.
SECTION: Working protocol
2. The user must first wash the specified quantity of magnetic beads in 1000µl buffer solution (PBS). For this purpose, 1000µl buffer solution is added to the beads, the tube is placed in the magnetic rack and the liquid is pipetted off after 2 minutes while the magnetic beads remain in the tube. Resuspend the beads in corresponding amount of buffer solution (40µl).
SECTION: Working protocol
4. Leave the mixture at room temperature for 30 minutes in dark. Shake occasionally.
SECTION: Working protocol
5. Place the tube in the Genekam magnetic rack for 3 minutes. During this step, the magnetic beads are attracted towards the magnet.
SECTION: Working protocol
6. Remove the liquid with a pipette tip carefully without distrubing the magnetic pellet and collect it in a new tube.
SECTION: Working protocol
7.  A test should be carried out to check whether the endotoxin has been extracted from the liquid. It is recommended. 
SECTION: Working protocol
8.  Used magnetic beads can be regenerated and reused.
SECTION: Working protocol
3. This protocol is for 1 ml of the solution, from which user wants remove the endotoxins. Take 1 ml of the solution and add 40µl of the magnetic beads to this.
SECTION: Working protocol
9.
TITLE: Tissue Punching Protocol
AUTHORS: McKenzie Pavlich, Lani Tieu, Brent Boomhower, Lisa Maturin, Giordano De Guglielmo, Olivier    George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SOP for brain punching for the Biobanks</div></div>

[STEPS]
?. [Beginning Prep]
Take brain out of -80° freezer and leave in -20 ° freezer overnight.
?. [Beginning Prep]
Make sure the cryostat is at -14° and the stage matches the cryostat temperature. If you plan on leaving the lid to the cryostat open during punching, you may want to turn the temperature a few degrees colder as the temperature warms up with the lid open.
?. [Beginning Prep]
Turn on cryostat light.
?. [Beginning Prep]
Get dry ice for tubes and crush it to fill a tube holder.
?. [Beginning Prep]
Label tubes for all desired regions.
?. [Beginning Prep]
Place blade in cryostat and make sure there is a good blade/plate ratio.
?. [Beginning Prep]
Find punching tools and place them nearby in crushed dry ice.
?. [Clean Up]
Remove blade.
?. [Clean Up]
Clear away discarded tissue sections.
?. [Clean Up]
Clean all chucks used.
?. [Clean Up]
Turn off cryostat light and set  cryostat back to desired temperature.
?. [Clean Up]
Return the stage to the home position.
?. [Clean Up]
Put all tools away.
?. [Clean Up]
Clean surrounding area.
?. [Punching]
When you are finished with punching, place all tubes with tissue in the -80° freezer
?. [Punching]
Make tally marks as you complete punches for each region.
?. [Punching]
Place punching tool in tube that has a label that matches the punched region. Leave in tube for ~10-15 seconds and then punch the tissue out with punching rod. If you do not see the pellet drop, it could have melted to the side of the tool. Ensure the punch is frozen and collected at the bottom of the tube before moving on.
?. [Punching]
Cut the brain slice at 500um and take punches that correspond with your markings.
?. [Punching]
Use the punching tools to make a mark where you would like to punch. Please see attached images for regions collected.
?. [Punching]
When you start to see your region of interest, switch the cutting thickness to 500um.
?. [Punching]
Begin cutting the brain at 50-100um thickness until you get to region of interest. It is OK to adjust stage during this process to make sure you are getting a full slice of the brain.
?. [Punching]
Once the glue has been added and is completely frozen place chuck on the stage and adjust the brain to correct for any slanting that occurred while cutting the cerebellum.
?. [Punching]
Add layers of OCT compound glue until the glue is over the weak joint point where the cerebellum connects. When adding additional layers of glue, make sure that the previous layer has frozen completely before adding the next layer.
?. [Punching]
Cut off 1/3 of cerebellum to create a flat surface for the brain to stand on.
?. [Punching]
When you are ready to begin, move brain from -20° freezer to cryostat. Let sit for ~5 min.
?. [Punching]
?. [Punching]
ALWAYS PLACE THE PUNCHING TOOLS BACK IN DRY ICE BETWEEN PUNCHES
?. [Beginning Prep]
Make sure that the cryostat blade is sharp (must be sharpened at least every 6-12 months)
TITLE: Aggregating expert-elicited data for prediction: A scoping review of statistical methods, experiments, and applications
AUTHORS: Thomas McAndrew, Nutcha Wattanachit, G. Casey Gibson, Nicholas G. Reich

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Combining forecasts from experts, compared to a statistical model, is often more accurate when data is sparse or prone to revision. Mathematical approaches for combining forecasts span several disciplines. Economics, marketing, population dynamics, and politics all rely on expert intuition to make predictions about the future. Because expert-elicited combination forecasting is spread across different domains, researchers use a diverse set of definitions to describe the same concepts. The diversity of applications also means methodological approaches are developed in parallel rather than building on one another. Our scoping review aims to unify these varied definitions and methodological approaches, map key terminology, and summarize applications of combination forecasting, experimental designs, and methods to mathematically aggregate expert-elicited data. </div><div class = "text-block">We will collect articles related to methods for aggregating expert-elicited forecasts from the Web of Science database. An article is defined as in-scope if it describes eliciting expert judgments and combining them to make a prediction about a natural phenomena or future event. Articles will be considered in-scope if two committee members read the article and agree it meets the above criteria. </div><div class = "text-block">From the final set of in-scope articles, we will extract: the application, experimental design, metrics used to compare forecasts, and the mathematical model used to combine forecasts. Common terminology will be consolidated and discussed. We will also ask of each article a pre-specified set of questions related to combination forecasting.</div><div class = "text-block">This protocol prespecifies all criteria and steps for conducting a scoping review on methods for aggregating expert-elicited predictions. All aspects of this protocol were reviewed and agreed on by the coauthors, and any significant deviations from this protocol will be recorded and an amended protocol submitted. This scoping review---selecting articles in a rigorous, prespecified, and unbiased way---aims to map, classify, and summarize all aspects of literature that aggregates expert-elicited predictions.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Computer-Aided Nodule Assessment and Risk Yield (CANARY) is a novel computed tomography (CT) tool developed at Mayo Clinic (Rochester, MN) that characterizes early lung adenocarcinoma by detecting nine distinct voxel classes, representing a spectrum of lepidic to invasive growth, within an adenocarcinoma. CANARY characterization has been shown to correlate with ADC histology and patient outcomes.</p>
<p> </p>
<p>This protocol provides basic instructions for segmentation of lung adenocarcinoma on CT imaging. CANARY has been validated in lung adenocarcinomas less than 3cm in diameter</p>

[STEPS]
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TITLE: Sample collection, embedding and freezing tissue
AUTHORS: Paul PN Norton, Emily Stephenson, Keval Sidhpura, Chloe Admane, Mohi Miah

[DESCRIPTION]
Sample collection, embedding and freezing paediatric skin tissue

[STEPS]
SECTION: Day 1 - Sample collection
1. Take 6mm punch biopsy of skin and place in a vial of Hypothermosol that has been pre-chilled at 4oC. Record time of biopsy.
SECTION: Day 1 - Sample collection
2. Proceed to embed and freeze the tissue as soon as possible, no longer than 24 hours later, ensuring that the sample is kept in Hypothermosol at 4oC in the mean time. Record time of freezing.
SECTION: Day 1 - Embedding
3. Prepare an isopentane-dry ice slurry. Place a small metal box onto dry ice, and fill with isopentane to a depth of several centimetres. Slowly add dry ice to the isopentane. 
SECTION: Day 1 - Embedding
4. Label cryomoulds with both sample and orientation information; it is recommended that at least the cutting face is noted on the cryomould. Half-fill cryomoulds with OCT then leave for at least several minutes; this will minimise bubbles.
SECTION: Day 1 - Embedding
5. Process tissues one at a time.
SECTION: Day 1 - Embedding
6. Prior to embedding and freezing, it is recommended that tissues are examined using a dissection microscope. 
SECTION: Day 1 - Embedding
7. Following all necessary examination and dissection, use blotting filter paper to quickly and gently remove any excess Hypothermosol.
SECTION: Day 1 - Freezing
8. Using spatula, transfer the tissue into a cryomould pre-filled with OCT. For skin tissue, place the sample with the epidermis facing down, and make a note of the surface needed to cut to ensure that all skin layers are sectioned
SECTION: Day 1 - Freezing
9. Carefully manipulate the orientation of the material to make sure the skin surface is at the bottom of the mould, but not touching the bottom if possible. Fill the rest of the mould with OCT so the sample is covered.
SECTION: Day 1 - Freezing
10. Use a pair of forceps to place the cryomould into the isopentane-dry ice slurry, keeping it level during movement to prevent loss of OCT and sample movement.
SECTION: Day 1 - Freezing
11. Rest cryomould immersed in the top of the isopentane liquid until the OCT has started to freeze
SECTION: Day 1 - Freezing
12. Once completely frozen, lift the cryomould out of the isopentane-dry ice slurry, and onto fresh dry ice, to allow excess isopentane to drain and evaporate. 
SECTION: Day 1 - Freezing
13. Wrap each sample, frozen in individual cryomoulds, in aluminium foil. Carefully label the outside of the foil prior to wrapping around each sample.
SECTION: Day 1 - Freezing
14. Transfer samples to a pre-cooled cryobox and store at -80°C.
TITLE: PCR Reaction for Experiments
AUTHORS: Kenneth Schackart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to run nucleic acid amplification using the Thermo scientific PCR Master Mix kit.</div><div class = "text-block">Each reaction produces 50 μL.</div><div class = "text-block"><span>For the original protocol, look at: </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Prepare Work Area]
Spray entire work area with 70% EtOH including pipettes, tip holder used for holding PCR tubes, and work surface. Wipe with a paper towel.
?. [Prepare Work Area]
Spray entire work area with RNAway.
?. [Gather Materials]
Take styrofoam container to Marley 527 (directly across from Marley 509) and fill halfway with ice.
?. [Gather Materials]
Set PCR tube holder on ice, and allow to cool for .
?. [Gather Materials]
Transfer Master Mix, primers, RNAse free water, and target tubes from freezer to PCR tube holder on ice.
?. [Gather Materials]
Allow reagents to thaw on ice
?. [Gather Materials]
Carefully obtain (2) 0.2 mL PCR tubes. Label one with "NTC" and the other "TARG". These will be your reaction vessels.
To avoid contamination when grabbing PCR tubes, only touch the outside of tubes. Avoid touching the inside of the caps of other tubes in this process. This is critical.
?. [Prepare Reaction]
Add the following to your two tubes:  ABC1TargetNTC2PCR Master Mix25 μL25 μL3Primer Mixture (25 μM)1 μL1 μL4Target (2.4×10^6 copies/μL)1 μL-5Water23 μL24 μL6Total50 μL50 μL
ABC1TargetNTC2PCR Master Mix25 μL25 μL3Primer Mixture (25 μM)1 μL1 μL4Target (2.4×10^6 copies/μL)1 μL-5Water23 μL24 μL6Total50 μL50 μL
?. [Gather Materials]
Vortex mix all reagents for approximately .
?. [Prepare Reaction]
Vortex mix the reaction mixture.
?. [Prepare Reaction]
Spin down reaction mixture.
?. [Run PCR Reaction]
Place reaction vessels into thermocycler.
?. [Run PCR Reaction]
Turn on thermocycler
?. [Run PCR Reaction]
Hit PROCEED to select a reaction cycle.
?. [Run PCR Reaction]
Scroll using the '' keys to get to EPCR.Begin using the following program:  ABCD1StepTemperatureTimeNumber Cycles2Denaturation95ºC3 min13Denaturation95ºC30 s304Annealing58ºC30 s5Extension72ºC60 s6Final Extension72ºC10 min1
ABCD1StepTemperatureTimeNumber Cycles2Denaturation95ºC3 min13Denaturation95ºC30 s304Annealing58ºC30 s5Extension72ºC60 s6Final Extension72ºC10 min1
?. [Run PCR Reaction]
Press PROCEED to begin
?. [Gather Materials]
Spin down all reagents for approximately .
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Long-standing questions in marine viral ecology are centered on understanding how viral assemblages change along gradients in space and time. However, investigating these fundamental ecological questions has been challenging due to incomplete representation of naturally occurring viral diversity in single gene- or morphology-based studies and an inability to identify up to 90% of reads in viral metagenomes (viromes).  In this protocol, I describe how to use an annotation- and assembly-free strategy for comparative metagenomics that combines shared k-mer and social network analyses (regression modeling). This robust statistical framework enables visualization of complex sample networks and determination of ecological factors driving community structure.  This tutorial describes a protocol to reproduce work from the Pacific Ocean virome comprised of 32 viromes from diverse sites in the Pacific Ocean.<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/b6ci6d.jpg" alt="" data-src="https://s3.amazonaws.com/pr-journal/b6bi6d.jpg" data-ofn="F1.large.jpg" /><br /> "Modeling ecological drivers in marine viral communities using comparative metagenomics and network analyses" (July 7, 2014, doi: 10.1073/pnas.1319778111, PNAS July 22, 2014 vol. 111 no. 29 10714-10719)<br /><br />Code is freely available at <a href="https://github.com/hurwitzlab/docker-fizkin" target="_blank">Github</a>.<br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br /><br />

[STEPS]
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