eamag/protocols-with-steps huggingface dataset:
TITLE: ActA purification protocol
AUTHORS: Sharifah Albraiki

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

[STEPS]
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When I did the gel-filtration step I lost all my protein.
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?. [Buffers]
Lysis Buffer:20mM MOPS100mM KCl5mM Imidazole     pH 7Wash Buffer:20mM MOPS100mM KCl20mM Imidazole     pH 7Elution Buffer:20mM MOPS100mM KCl250mM Imidazole     pH 7Storage Buffer:20mM HEPES100 NaCl               pH 7.5
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol outlines the Bray-Curtis dissimilarity NMDS ordination and significance analysis of our manuscript. Here we will look at the clustering of the virome samples using our reference independent OTU table based on contig hits. We will compare skin environments (sebaceous, etc), occlusion status, and time. We also look at the difference between the background controls and the rest of the samples. Based on the methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.<br /><br /><br />

[BEFORE_START]
Supplemental information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<span class="identifier">sessionInfo</span><span class="paren">(</span><span class="paren">)<br /><br />## R version 3.2.0 (2015-04-16) <br />## Platform: x86_64-apple-darwin13.4.0 (64-bit) <br />## Running under: OS X 10.10.4 (Yosemite) <br />## ## locale: <br />## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 <br />## <br />## attached base packages: <br />## [1] stats graphics grDevices utils datasets methods base <br />## <br />## loaded via a namespace (and not attached): <br />## [1] magrittr_1.5    formatR_1.2    tools_3.2.0    htmltools_0.2.6 <br />## [5] yaml_2.1.13    stringi_0.4-1    rmarkdown_0.7    knitr_1.10.5 <br />## [9] stringr_1.0.0    digest_0.6.8    evaluate_0.7</span>

[STEPS]
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TITLE: Very New York Behavioural Analysis Protocol v2.3
AUTHORS: Gabriel  Frazer-McKee, Bruno Courbon

[DESCRIPTION]
This protocol contains descriptions of the constructional and semantico-pragmatic variables assessed while annotating the “very New York” dataset in our 2022 paper published in the journal Signifiances. It should be noted that many of these variables were not considered in the article owing to either time-constraints, space-constraints or because the variable was ultimately discarded.

[STEPS]
TITLE: Computer-Aided Drug Discovery
AUTHORS: BOC  Sciences

[DESCRIPTION]
Computer-aided drug design (CADD) is a widely used technology using computational tools and resources for the storage, management, analysis and modeling of compounds. It relies on digital repositories for the study of designing compounds with physicochemical characteristics, predicting whether a given molecule will be combined with the target, and if so how strongly. Computer-based methods can help us to search new hits in drug discovery, screening many irrelevant compounds at the same time and study the structure-activity relationship of drug molecules.

[STEPS]
TITLE: Buck Institute Morphology H &amp; E staining protocol
AUTHORS: sbreslin

[DESCRIPTION]
 Histological Stain for FFPE slides
RESULTS:  Nuclei-blue; Cytoplasm, red blood cells and connective tissue-shades of pink

[BEFORE_START]
Slides can be placed in 60 C oven prior to deparaffinization. 

[STEPS]
SECTION: Hematoxylin and Eosin Staining
1. Deparaffinization and rehydration: 2 x xylene 7 min, 100% ethanol 4 min, 95% 4 min, 80% 4 min, 70% 4 min, bring to water.
SECTION: Hematoxylin and Eosin Staining
2. Hematoxylin staining for 30 seconds - 4 min. using filtered dye (usually 2 mins) - Modified Mayes’s Hematoxylin HXMMHPT StatLab Medical Products
SECTION: Hematoxylin and Eosin Staining
3. Rinse in ddH20 x2
SECTION: Hematoxylin and Eosin Staining
4. 10% acetic acid rinse (acid into water) – 1 min
SECTION: Hematoxylin and Eosin Staining
5. rinse in ddH20 x2
SECTION: Hematoxylin and Eosin Staining
6. TBS (1x) rinse or Scott’s Tap Water (1L + 3.5g Sodium Bicarb + 20g Magnesium Sulfate pH 8.0)  - 2 mins
SECTION: Hematoxylin and Eosin Staining
7. Rinse in ddH20
SECTION: Hematoxylin and Eosin Staining
8. Rinse in 70% ETOH - 1 min
SECTION: Hematoxylin and Eosin Staining
9. Stain Eosin for 0.5 to 2 minutes (keep an eye on intensity of pink) – Eosin Y Stain STE0150 StatLab Medical Products
SECTION: Hematoxylin and Eosin Staining
10. Rinse in 90% EtOH
SECTION: Hematoxylin and Eosin Staining
11.  Rinse in 2 x 95% EtOH, 2 x 100% EtOH,  xylene
SECTION: Hematoxylin and Eosin Staining
12. Coverslip with Permount Mounting Medium EMS #17986-01
TITLE: High Dose STZ Induction Protocol
AUTHORS: Frank Brosius

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block"><span>This protocol is used by DiaComp members to induce diabetes in a number of the animal models developed by the consortium. STZ is toxic to the insulin- producing beta cells of the pancreas and used to induce a diabetes similar to a type I diabetic (</span><a href="https://www.sciencedirect.com/science/article/pii/S1383574202000443" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Reference</span></a><span>). Some reports suggest cellular toxicity outside of the pancreas. STZ also exhibits broad spectrum antibacterial properties and alters the gut microbiota. Please ensure that appropriate controls are included in all studies and complementary models considered (e.g. the</span><span style = "font-weight:bold;"> Ins2</span><span style = "vertical-align:super;">Akita</span><span> mouse).</span></div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complications:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhwp4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhwq4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhwr4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhws4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhwt4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhwu4kcn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bhwv4kcn.jpg" /></div></div>

[STEPS]
?. Prepare the buffers and solutions as described below. Please note that the STZ- Na-Citrate solution should be prepared immediately prior to injection so as to avoid degradation of the STZ.
?. Mice should be fasted prior to injection; four hours is usually sufficient.
?. Place one mouse in the Isoflurane drop jar, according to your local IACUC anesthesia standards.
?. Remove mouse when breathing has slowed and animal is anesthetized
?. Inject appropriate amount of the STZ solution IP so the final dosage is 150 mg/kg mouse
?. Allow mouse to awaken and place back in cage
?. Repeat procedure for each animal; keep in mind that Isoflurane will need to be re- loaded after every third or fourth animal for proper anesthesia.
?. Each mouse should be given one injection.
?. Supply mice with 10% sucrose water overnight to avoid sudden hypoglycemia post-injection.
?. Mice should be tested for sufficient levels of hyperglycemia two days after injection and 4 weeks post-injection. Mice should be severely diabetic.
TITLE: The Podiatrists in Australia: Investigating Graduate Employment (PAIGE) Study
AUTHORS: Cylie  Williams, Anna Couch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">The Podiatrists in Australia: Investigating Graduate Employment (PAIGE) study provides a systematic and rigorous evidence-base for rural podiatric workforce policy development, by developing a database modelled on the highly successful Medicine in Australia, Balancing Employment and Life (MABEL) longitudinal panel survey of Australian doctors. Analysis of data from waves 1 and 2 of this database, and concurrently collected qualitative data, address two key research questions about work location choice decisions and retention of podiatrists in location and the profession.  </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
TITLE: Sholl analysis 
AUTHORS: mariangela.massarocenere

[DESCRIPTION]
Protocol for Sholl analysis by Neurolucida software 

[STEPS]
SECTION: cell body and branching tracing
1. Select the center for the analysis and the structures or markers to be compared
SECTION: cell body and branching tracing
1.1. Select one cell body that you want to use as the center for the analysis
SECTION: cell body and branching tracing
1.2. Select one or more process/group of processes, puncta, and/or markers to be analyzed in relation to the cell body 
SECTION: Analysis
3. Go to Analyze-> Neuron data -> Spatial and click SHOLL ANALYSIS
SECTION: Analysis
3.1. Spheres: Choose the starting radius (0µm) and radius increment (10µm) for the spheres
SECTION: Analysis results
5.  Radius: Each radius corresponds to a shell. The number shown is the position of the shell in µm from the designated center point
Intersections: Number of intersections between process and sphere at the given radius
Length: Total length in µm of all processes passing through a shell
Nodes: Total number of nodes in the shell
Endings: Total number of endings in the shell
SECTION: cell body and branching tracing
2. Trace cell body contour and branching from the cell body in continuous mode
SECTION: Analysis
4. Analyze at least fifteen cells per animal (randomly selected from 3 serial coronal sections/animal) and subsequently average all data for each animal 
SECTION: Analysis
3.2. Analyses: Choose the analyses that you want to include (e.g branching)
TITLE: Serial Alberta Stroke Program Early CT Score to Predict Stroke-Associated Pneumonia after Thrombolysis in Patients with Acute Ischemic Stroke
AUTHORS: Sarawut Krongsut, Atiwat Soontornpun, Niyada Anusasnee

[DESCRIPTION]
Objectives: To investigate the performance of serial Alberta Stroke Program Early CT Score (ASPECTS)
assessment for predicting stroke-associated pneumonia (SAP) in patients with thrombolyzed
acute anterior circulation ischemic stroke (AACIS).
Materials and Methods: A retrospective
observational cohort study of adult patients with thrombolyzed AACIS was
conducted. Baseline and 24-hour ASPECTS using non-contrast computed tomography (NCCT), complications of stroke, including SAP and swallowing dysfunction
using the Modified Water Swallowing test, were collected. Receiver operating
characteristic curves using binary logistic regression and a multivariable
logistic regression model were generated to compare the predictive performance.


[STEPS]
1. Serial Alberta Stroke
Program Early CT Score to Predict Stroke-Associated Pneumonia after Thrombolysis
in Patients with Acute Ischemic Stroke
TITLE: Immunohistology
AUTHORS: Mai-Anh Vu, mwhowe

[DESCRIPTION]
We have developed a new micro-fiber array approach capable of chronically measuring and optogenetically manipulating local dynamics across over 100 targeted locations simultaneously in head-fixed and freely moving mice, enabling investigation of cell-type and neurotransmitter-specific signals over arbitrary 3-D volumes . This protocol includes the immunohistology steps we used to confirm viral expression, and/or stain for neurons after our micro-CT scanning and contrast agent soaking procedure.

[STEPS]
SECTION: Removing Lugol&#x27;s contrast agent from tissue
1. In preparation for immunohistology, Lugol’s solution was removed from brain tissue after CT imaging by soaking the implanted brains in a solution of 5% sodium thiosulfate (STS, Carolina) in 1% PBS for 4-6 days (Hopkins et al., 2015).
SECTION: Removing Lugol&#x27;s contrast agent from tissue
2. The implant was then removed, the brain returned to the STS solution for one hour.

SECTION: Slicing
4. Once the brains sank, coronal sections (50 µm) were sliced with a cryostat (Leica CM3050 S) and transferred to PBS for storage.
SECTION: Immunostaining
5. To stain for dLight1.3b or ChR2 expression:

SECTION: Immunostaining
5.2. Treated sections were then incubated for 24-48 hours at 4°C in 5% NGS, 0.2% triton, and a GFP primary antibody (chicken polyclonal antibody; 1:1000, ThermoFisher Scientific, No. A10262).
SECTION: Immunostaining
5.3. Slices were then washed, and incubated for 2 hours at room temperature in 5% NGS, 0.2% PBST, and an Alexa 488 secondary antibody (goat anti-chicken; 1:200, ThermoFisher Scientific, No.A-11039).
SECTION: Immunostaining
6. For neuronal staining:

SECTION: Immunostaining
6.2. Treated sections were then incubated for 24-48 hours at 4°C in 5% BSA, 0.2% triton, and a NeuN primary antibody (guinea pig antibody; 1:500, Synaptic Systems, 266 004).
SECTION: Immunostaining
6.3. Slices were then washed, and incubated for 2 hours at room temperature in 5% BSA, 0.2% PBST, and an Alexa 647 secondary antibody (goat anti-guinea pig; 1:200, ThermoFisher Scientific, No. A-21450).
SECTION: Mounting and imaging
7. Finally, stained sections were mounted onto glass slides using Vectashield Mounting Medium with DAPI (Vector Laboratories, H-2000).
SECTION: Mounting and imaging
8. Confocal images were acquired with a Zeiss LSM 800.
SECTION: Slicing
3. The brains were then moved to a solution of 30-40% sucrose in PBS.
SECTION: Immunostaining
5.1. Sections were then blocked and treated in a 5% normal goat serum (NGS) and 0.2% PBS triton (Sigma Aldrich) solution (PBST).
SECTION: Immunostaining
6.1. Sections were then blocked and treated in a 5% bovine serum albumin (BSA) and 0.2% PBS triton (Sigma Aldrich) solution (PBST).
TITLE: Preparation of soluble and insoluble mitochondrial protein fractions for immunoblotting
AUTHORS: Louise Uoselis

[DESCRIPTION]
Preparation of soluble and insoluble mitochondrial protein fractions from HeLa cells for immunoblotting.

[STEPS]
1. 
 
Thaw mitochondrial stocks on ice, and take two aliquots of 15 µg of mitochondria each for each sample (one aliquot will be the ‘total’ sample fraction, and one aliquot will represent the ‘fractionation’ sample).  

2. Centrifuge samples at 10000 rcf for 10 min at 4 °C, and carefully aspirate the supernatant.

3. Add 15 µL of ice cold 0.5% v/v TX-100 in PBS, vortex each sample for ~5 seconds, and leave samples to incubate on ice for 15 min. At the conclusion of the 15 min lysis incubation, vortex each sample for ~5 sec again. 

4. Set aside one fraction from each sample for processing downstream (referred to as the ‘total’ fraction). 
5. Centrifuge the remaining tube of each sample at 12000 rcf for 10 min at 4 °C

6. Using a pipette, carefully remove 14.24 µL of the supernatant (representing 95% of the lysis volume) and place this sample into a clean microfuge tube on ice, being sure not to disturb any pellets that have formed on the bottom of the tube. The volume removed represents the ‘soluble’ protein fraction. The tube containing the pellet represents the ‘insoluble’ protein fraction. 

7. Add 14.24 µL of 0.5% TX-100 in PBS to each insoluble protein fraction by pipetting on the side of the tube. Gently flick each tube to rinse the sides of the tube. 

8. Centrifuge the insoluble protein fractions at 12000 rcf for 10 min at 4 °C.

9. Using a pipette, carefully remove 14.24 µL of the supernatant from the insoluble protein fraction, and place into a clean microfuge tube (which will function as the waste collection tube for all samples).

10. Repeat steps 7 – 9, which will total 2 washes
11. Repeat step 7. You should now have three tubes for each sample (representing the total, soluble and insoluble protein fractions), and one waste tube. 
12. Bring all samples to room temperature. To the total and insoluble samples, add 5 µL of 4x SDS loading dye (4x: 20% w/v SDS, 400 mM DTT, 40% v/v glycerol, 200 mM Tris-Cl pH 6.8). To the soluble fraction, add 4.75 µL of 4x SDS loading dye. 
 


13. Vortex each sample for ~3 seconds
14. Boil all samples at 99 °C for 10 min shaking at maximum speed.

15. Let samples cool to Room temperature, and centrifuge quickly to bring all liquid to the bottom of the tubes. Vortex the samples again for ~3 sec to ensure homogeneity. 

16. Sonicate all samples in a waterbath sonicator set to 21 °C  water temperature for 2 min. 

17. Remove all samples from the sonicator, vortex samples for ~3 sec to mix. 
18. Load 19 µL from each sample on an SDS-PAGE gel and proceed to run and transfer the gel as per a standard immunoblotting protocol.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The Sequence Read Archive (SRA) is a database for biological sequence data and is maintained by the National Center for Biotechnology Information (NCBI). Sequence files can be obtained by using the SRA Toolkit. This protocol provides the steps necessary to use the SRA toolkit to get sequence data in fastq format. </p>

[BEFORE_START]
<p>Login to the UA hpc. This protocol will begin in your home directory. </p>

[GUIDELINES]
<p><a href="https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=toolkit_doc" target="_blank">SRA Toolkit Documentation</a></p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Preparation of Zymos<em>eptoria tritici </em>isolates for extraction of chromosomes via pulsed-field electrophoresis</p>
<p> </p>
<p>Pat Martinez 1990 (Bruce McDonald lab) (Simplified from Cooley, et al, Curr. Genet. 13:383-389.)</p>

[STEPS]
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TITLE: Working Reverse Primer Plate(s)
AUTHORS: Allyson Hirsch, George  Testo

[DESCRIPTION]
Stock primers should not be directly used in a PCR because they are concentrated. Working from one tube is a bad idea, and thus it only takes a small amount of contamination to render your primers ineffective. For this reason, it is best practice to create working solutions that are of lower concentrations. The concentration of choice for a working primer solution is user-dependent. However, 10uM is used for this protocol.

[BEFORE_START]
Make sure you work in a DNA-free environment. Preferably in a PCR preparation hood. This is to avoid contamination of your stock and working primer solutions.


Use PCR-grade water (DNase- and RNase-free) to reconstitute and dilute your primers.


Use filter pipette tips to prevent contamination via pipetting.

[STEPS]
SECTION: Preparing PCR Hood
1. Decontaminate a PCR hood with DNA Away and Ethanol.
SECTION: Preparing PCR Hood
2. Place materials inside of the PCR Hood (refer to materials section).
SECTION: Preparing PCR Hood
3. Turn on the UV setting for at least 30 min.

SECTION: Preparing Skirted Plates
4. Temporary seal 6 96-well skirted plates and label each “Reverse Primer Plate ___ Initials/ Date.”
SECTION: Preparing Skirted Plates
5. Add 90 µL of molecular grade water (poured into a 50 mL conical) to each well with a 200uL single-channel pipette. Make sure to get a new tip for each subsequent well (this techelps prevent contamination).

SECTION: Preparing Skirted Plates
6. After completing 3 primer plates, retrieve the 100 micromolar (µM) stock from the freezer and let it thaw.

SECTION: Preparing Skirted Plates
7. Finish the remaining 3 primer plates.
SECTION: Preparing Skirted Plates
7.1. Temporary seal each primer plate after loading with molecular grade water.
SECTION: Preparing Skirted Plates
8. Once the EMP 100 micromolar (µM) stock has thawed, spin each plate down for 1 min at full speed.

SECTION: Adding EMP Primer(s)
9. Add 10 µL from each well from the EMP 100 micromolar (µM) primer plate using a 10uL multichannel pipette, and pipetting to corresponding well(s) on the 10 micromolar (µM) primer plate loaded with water.

 


SECTION: Adding EMP Primer(s)
10. Once the 10 micromolar (µM) primer plate stock has been made, seal with a foil freezer seal and store in the -20 °C freezer.
TITLE: Core Protocol for Serial Imaging of Fluorescently-Labeled Mouse Brain on the TissueCyte 1000 System
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the instructions for setup and scanning of fluorescently-labeled tissue sections mounted in agarose and attached to a slide using the TissueCyte 1000 block face 2-photon system.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: 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.</span></div></div>

[STEPS]
TITLE: BD FACS Aria II Shutdown
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol provides step by step instructions for shutting down the BD FACS Aria II and cleaning it of potentially hazardous materials.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: 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.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is for extracting RNA from 25 mm filters and can be used with filters stored in RNAlater for preservation. This protocol has been tested with 0.22 μm pore size Durapore filters. Custom synthesized RNA transcript standards are added at the time of extraction and are recovered post-sequencing for quantitative metatranscriptome analysis (Satinsky et al., 2013).

[BEFORE_START]
Have the following on hand in addition to materials: forceps, 2.0 and 1.5 ml RNase free tubes, temperature controlled microcentrifuge, vortex with 2.0 ml adaptors, 21g1 needls, and 3 ml syringes.

[GUIDELINES]
<span style="text-decoration: underline;">Reference:</span><br /><br />Satinsky BM, Gifford SM, Crump BC, Moran MA (2013) Use of internal standards for quantitative metatranscriptome and metagenome analysis. Meth Enzymol, ed DeLong EF (Academic, Burlington, MA), Vol 531, pp 237–250

[STEPS]
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TITLE: 2021 Featured Protocols
AUTHORS: protocols.io team

[DESCRIPTION]
This is a growing collection that includes all protocols that were featured in 2021 on our welcome page and on social media (Twitter, Facebook). 

Every week, we feature 3 new protocols that are particularly fantastic examples of reproducible and detailed method sharing. (Please note that there are many weeks where we would love to highlight dozens of public protocols instead of three!)


[STEPS]