eamag/protocols-with-steps huggingface dataset:
TITLE: Experiment Data Depot (EDD) Study Creation
AUTHORS: Jennifer Gin, Christopher J Petzold

[DESCRIPTION]
This protocol details how to create studies in the Experiment Data Depot. Data can be shared with collaborators via exported CSV files or through the REST API.

For more information about the EDD visit: Experiment Data Depot Google Site.

Citation: 
Morrell, William C., et al. "The experiment data depot: a web-based software tool for biological experimental data storage, sharing, and visualization." ACS synthetic biology 6.12 (2017): 2248-2259. (link to publication)

Example Links:
https://public-edd.agilebiofoundry.org/
https://public-edd.agilebiofoundry.org/s/example-data-quality-study-2/overview/



[GUIDELINES]
Line Name:  Line name should only have dash (-), underscore (_), or space ( ).

Part ID:  Make sure that your ICE registry part ID number is accessible to collaborators and that it is a strain, not a plasmid.



[STEPS]
1. Go to the appropriate EDD for your project (e.g., public-edd.agilebiofoundry.org).  Login with your  username and credentials.

 

 
 

2. Click on the New Study button on the top right.
 
 


3. In the Add New Study pop-up window, type in your Study Name and Contact.  Click Add Study button.

 

 


4. On the Overview page, click on the Example File link in the center box to download a sample experiment description example file.  Use this as a template file to fill out your sample experimental conditions.  Or, you can click on the Enter Manually button to type in your sample experimental conditions, one by one.

 

 

 
 
  
 


  





5. Drag-and-drop the Sample Experiment Description Example File into the dropzone on the Overview page.

 



8. Once you have acquired the data, go to the EDD Data Import protocol to import data into the EDD study.
6. Click on Permissions to allow collaborators to Read or Write data in the study.


 


7. Click on Attachments to attach files to the study.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The diverse applications of mutant and recombinant adenoviruses demand facile methods for their generation. This protocol allows the user to rapidly assemble customized adenoviral genomes from high-copy plasmids in a two-step isothermal reaction. Following <em>in vitro</em> assembly, full-length genomes can be directly packaged in HEK293 cells. Crude preparations of infectious virus can be prepared in less than one week.</p>

[STEPS]
TITLE: Open Isothermal Platform Protocal
AUTHORS: Andrew Buultjens, Koen  Vandelannoote, Tim Stinear

[STEPS]
?. Place strips in FABL-8
?. Cap all wells
?. Add 5ml of positive control in well 8.
?. Add 5ml of H2O in negative control well 7.
?. Transfer 5ml of extracted RNA to pre-aliquoted LAMP mix. Mix by pipetting up and down 5 times.
?. Extract RNA as per dx.doi.org/10.17504/protocols.io.bkynkxve
?. LAMP reaction (can refer to N1-STOP-LAMP paper)Single reaction setup:   AB1ReagentVolume (ml)2Optigene Master Mix153N1 primer54Extracted RNA55Total25
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							To reduce pipetting error, Master Mix and N1 primer are premixed according to the number of reactions desired (allow for pipetting error by preparing enough for ~1.1x the number of reactions, rounded to the nearest whole number); 20ml of this mixture is then aliquoted per reaction.
AB1ReagentVolume (ml)2Optigene Master Mix153N1 primer54Extracted RNA55Total25
?. Run FABL-8 as per manual
TITLE: MALE CIRCUMCISION FOR PREVENTION OF HETEROSEXUAL TRANSMISSION OF HIV IN ADULT MALES IN SOWETO, what do indicators and incidence rates show?untitled protocol
AUTHORS: Hillary Mukudu, Neil Martinson, Benn Sartorius

[DESCRIPTION]
<p><strong>Background</strong></p>
<p>The biomedical prevention of HIV transmission that medical male circumcision confers was confirmed in a clinical trial setting and rolled out in VMMC programmes. Data assessing the effectiveness of this in a programmatic setting is not available. Concerns about possible risk compensation in circumcised males after circumcision have been raised.</p>
<p><strong>Aim</strong></p>
<p>To determine the effectiveness of medical male circumcision in prevention of HIV transmission and risk compensation</p>
<p><strong>Objectives</strong><br />• To determine the characteristics of HIV sero-negative males aged 18-40 years who seek circumcision.<br />• To determine the incidence of HIV in circumcised males aged between 18 -40 years of age in a mass male circumcision setting.<br />• To determine the risk factors associated with HIV sero-conversion among circumcised males aged 18-40 years.<br />• To determine the effect of circumcision on indicators of HIV transmission</p>
<p><strong>Methods</strong><br />The study will be a Cohort study design. Clients seeking circumcision will, upon providing consent, be enrolled in to the study. All will be provided with HIV counselling and testing as is currently the norm. A questionnaire will be administered by trained interviewers, prior to the circumcision. Both will be done on the same day. Two to seven days post circumcision they will be seen to check for any surgical complications as is currently being done in routine practice. There are follow-up visits that will be scheduled for 6 and 12 months post circumcisions. At these visits a questionnaire will be administered as well as an HIV test done. These will require that the participants come back to the clinic. To minimise loss to follow up subjects will be called by telephone or sent SMS reminders as to the days of follow up as well as a designated member of the study team will phone the subjects at least once a month.</p>

[STEPS]
TITLE: Proteomic Analysis of Human Ovarian Cortex and Medulla Secretome Using Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) Acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid Mass Spectrometer
AUTHORS: Joanna Bons, J P Rose, M A Watson, B Schilling

[DESCRIPTION]
Ovaries from human donors were cut into 3-5 mm sections. These sections were further processed into 500 μm slices containing cortex and medulla. The slices were then processed into pieces (1 mm x 1 mm x 500 µm), and cortex and medulla pieces were cultured separately as explants in static cultures. 
Explants were cultured and treated with either DMSO vehicle control or 0.1 µg/mL doxorubicin to induce
senescence. After 10 days, cortex and medulla explants were thoroughly washed with serum-free basal media and transferred to a clean plate with pre-equilibrated serum-free basal media and inserts. The conditioned media were collected after 24 hours for secretome proteomics profiling. 

The concentrated conditioned media was subjected to tryptic digestion using S-trap Spin columns. The reconstituted peptide elution was desalted with C18 hydrophilic-lipophilic balance (HLB) cartridges. The final reconstituted peptides were diluted with 2% ACN and 0.1% FA. Proteolytic peptide measurement was completed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) acquisition by Data-Independent Acquisition (DIA) on an Orbitrap Eclipse Tribrid mass spectrometer for peptide/protein identification and quantification.

[STEPS]
SECTION: Conditioned Media Concentration with Amicon Ultra Centrifugal Filters
1. dx.doi.org/10.17504/protocols.io.e6nvw1px7lmk/v1
SECTION: Protein Digestion with S-trap Spin Columns using Conditioned Concentrated Media
2. dx.doi.org/10.17504/protocols.io.x54v928eml3e/v1
SECTION: Proteolytic Peptide Desalting with C18 HLB Cartridges
3. dx.doi.org/10.17504/protocols.io.eq2lywdzpvx9/v1
SECTION: DIA Data Processing using Spectronaut/directDIA (Biognosys): Secretome Analysis
5. dx.doi.org/10.17504/protocols.io.q26g71rz3gwz/v1
SECTION: LC-MS/MS Acquisition by DIA on an Orbitrap Eclipse Tribrid Mass Spectrometer
4. dx.doi.org/10.17504/protocols.io.36wgq3m15lk5/v1
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a collection of Bioline protocols for the <span style="color: #000000; font-family: Arial; font-size: 13px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: left; text-indent: 0px; text-transform: none; white-space: pre-wrap; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; display: inline !important; float: none;">SensiFAST™ Kits. Please refer to the appropriate protocol, depending on your application.</span></p>

[STEPS]
?.
TITLE: Colorimetric determination of urea
AUTHORS: noah.langenfeld  , Laurenpayne  , Bruce  Bugbee

[DESCRIPTION]
This protocol measures the absorbance of urea in solution in complexation with diacetyl monoxime at 520 nm and is linearly proportional to concentration up to 5 mM urea.

[BEFORE_START]
Ensure a lab coat, lab goggles, and plastic gloves are worn throughout this assay.

[STEPS]
SECTION: Mixed Acid Reagent Preparation
1. 


Dissolve 2.5 mg  ferric chloride (97% purity) in 45 mL  deionized water in a 250 mL volumetric flask.  

SECTION: Mixed Acid Reagent Preparation
2. Add 80 µL of 14.8 Molarity (M) concentrated phosphoric acid (85 wt. % phosphoric acid in water).

SECTION: Mixed Acid Reagent Preparation
3. 
 
Prepare 4.7 Molarity (M) sulfuric acid by diluting 65.25 mL concentrated sulfuric acid (18 Molarity (M) , 98% purity) up to 250 mL with deionized water.

SECTION: Mixed Acid Reagent Preparation
4. Dilute ferric chloride, deionized water, and phosphoric acid mixture to 250 mL with 18 Molarity (M) sulfuric acid.

SECTION: Mixed Acid Reagent Preparation
5. Mix until dissolved. 
SECTION: Mixed Color Reagent Preparation
6. Add 20.9 mg  diacetyl monoxime (99% purity) and 52.4 mg thiosemicarbazide (99% purity) to a 250 mL volumetric flask.

SECTION: Mixed Color Reagent Preparation
7. Dilute to volume (250 mL) with deionized water. 

SECTION: Mixed Color Reagent Preparation
8. Mix until dissolved. 
SECTION: Urea Assay
9. Prepare known concentrations of urea at 0, 1, 2, 3, 4, and 5 mM for a calibration curve.

SECTION: Urea Assay
10. Fill a 600 mL beaker with 200 mL deionized water.

SECTION: Urea Assay
11. Place the 600 mL beaker on hot plate and bring to a boil.

SECTION: Urea Assay
12. To test a sample, aliquot 1 mL into a 20 mL glass test tube.

SECTION: Urea Assay
13. Add2 mL Mixed Acid Reagent.

SECTION: Urea Assay
14. Add 2 mL Mixed Color Reagent.    

SECTION: Urea Assay
15. Ensure contents of test tube are vigorously mixed by vortexing for at least 5 s .

SECTION: Urea Assay
16. Repeat steps 12-15 for each sample and standard to be analyzed.

SECTION: Urea Assay
17. Ensure test tubes are labeled and place in boiling water bath for 20 min .

SECTION: Urea Assay
18. Remove tubes from water bath and let cool for 10 min .

SECTION: Urea Assay
19. Fill plastic cuvette to line with cooled solution. 
SECTION: Urea Assay
20. Place sample cuvette in spectrophotometer and record absorbance at 520 nm. Blank against the standard solution with both reagents, but no urea.
SECTION: Urea Assay
21. Use the calibration curve to determine urea concentrations of unknown solutions.
TITLE: U Mass - Insulin tolerance test
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">
Insulin tolerance test measures systemic clearance of glucose following an intraperitoneal bolus injection of a physiological dose of insulin. This experiment measures insulin sensitivity in awake mice assuming that there are no alterations in the animal’s counterregulatory response. Insulin sensitivity is altered in obese mice.</div></div>

[STEPS]
?. Mice are fasted for 5 hours prior to the start of experiment.
?. Collect plasma sample (20 µl) before the start of experiment (basal-0 min) to measurebasal insulin and glucose levels.
?. Administer intraperitoneal injection of insulin (0.5 or 0.75 unit/kg body weight) using aninsulin syringe.
?. Collect plasma samples (10 µl) at 10, 20, 30, 60, 90 and 120 min for the measurement ofglucose concentrations.
?. For data analysis, plasma glucose levels vs. time after insulin injection are plotted, andarea-under-curve may be calculated to estimate insulin sensitivity.
TITLE: MAPS image analysis
AUTHORS: jessechao 

[STEPS]
?. [Setting up required environment and packages]
Navigate to https://github.com/jessecanada/MAPS, download the repository.
?. [Setting up required environment and packages]
The following commands assume that you are using conda (or Anaconda) to manage your python packages and that you are on a Unix type system (Linux or OSX).
?. [Setting up required environment and packages]
Use the following commands in terminal:Now you are ready to rock and roll. For additional details, see https://docs.conda.io/projects/conda/en/latest/user-guide/tasks/manage-environments.html
?. [Step 1 - image QC]
Open "MAPS_1_img-QC.ipynb" in Jupiter Notebook and follow the steps in the notebook to remove blurry images.
?. [Step 2 - cell detection]
Follow this quick start guide to build your first object detection model on Azure. (link: https://docs.microsoft.com/en-us/azure/cognitive-services/custom-vision-service/get-started-build-detector)Note that you can augment the training images you create on Azure. To do this, follow the optional training augmentation step below.Take note of the endpoint, training key, prediction key, resource ID and project ID.
?. [Step 2 - cell detection]
Open "MAPS_2_cell-detection.ipynb". We would recommend opening this notebook on Google Colab. Follow the steps in the notebook to crop out individual cells.
?. [Step 3 - phenotype discovery]
There are two options here. To use the method in Figure 5A, open "MAPS_3_conv_stacking.ipynb" and follow the instructions in the notebook to generate cell galleries. Alternatively, to use the method in Figure 5C (i.e. deep autoencoder), open "MAPS_3_autoencoder.ipynb" and follow the steps in the notebook. Again, we recommend opening the notebooks in Colab.
?. [Step 3 - phenotype discovery]
After generating cell galleries for wild type and a few variants, carefully inspect them to decide how many classes of localizations you need to classify.
?. [Step 4 - phenotype classification]
Gather the cropped individual cell images. Open "MAPS_4_phenotype_classification.ipynb" and follow the instructions in the notebook to make predictions and classify phenotypes. Again, we recommend opening this notebook on Colab. In the end you will get a .csv file containing predictions for each cell.Congratulations, you've made it to the end!
?. [Step 2 - cell detection]
(optional) Once you have labeled ~100 training images, you can perform training augmentation to boost the performance of your model. Note that it is always better to label more training images.Open "MAPS_2.1_training_augmentation.ipynb". We recommend opening this notebook on Google Colab so that you don't need to install additional packages
TITLE: Hydroxyl Radical-scavenging Activity
AUTHORS: Jing Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The scavenging of hydroxyl radicals were determined by Guo et al. The reactions were performed in 0.3 ml of 20 mM sodium salicylate, 2.0 ml of 1.5 nM FeSO</span><span style = "vertical-align:sub;">4</span><span>, 1.0 ml of samples, and 1.0 ml of 6 mM H</span><span style = "vertical-align:sub;">2</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">2</span><span>.The reaction mixture was incubated for 1 hat 37 °C. The absorbance was measured at 510 nm. The inhibition percent and 50% of absorbance(EC</span><span style = "vertical-align:sub;">50</span><span>)were calculated. </span></div></div>

[STEPS]
TITLE: Sigmoid fitting in Excel (Excel Solver Add-In)
AUTHORS: Kenji Ohgane

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol covers how to fit sigmoidal curve to data within Excel, and allows rapid estimation of EC50/IC50 values from experimental dose-response data. Although R or other specialized software is more suitable for detailed analyses, it is sometimes useful to perform sigmoidal fitting within Excel. Note that this is just a simplified version (only sigmoid fitting) of the protocol reported in Gerdi Kemmer & Sandro Keller (2010) Nat. Protocol 5: 267–281, and intended for routine laboratory use.</div></div>

[STEPS]
?. Download the following excel file. Copy the excel sheet into your excel file if needed.The file contains a sheet set up for sigmoidal fitting with an example data set, for which you can test the fitting procedure.
?. Open the excel file, and enter your data in the "x" and "y" column. Delete unnecessary data.
The data in the "x" column should be in decreasing or increasing order. Otherwise, the plot will not be correctly displayed.
?. Guess initial parameters for the sigmoid (minimum, max, n, and ec50), which can be easily guessed from the plot, and enter the estimated initial values into the cells (B2, B3, B4, B5).
If the initial guess is too far from the real values, the fitting algorithm may not result in reasonable fit.
?. Start Excel Solver by selecting "Solver" from "Tools" menu.
?. Set "Target cells" to "$B$7", which contains SSR (Sum of Squared Residuals).
?. Set "Changing cells" to the cells that contains initial values ("$B$2:$B$5").
?. Add constraints if needed. We recommend to add "ec_50 >= 0.000001" etc., to avoid EC50 becoming negative values or zero.
?. Uncheck "Assume non-negative", if checked.
?. Basically, no need to change the other options.
?. Click "Solve" to run the Solver, wait several seconds. Close the Solver window when the calculation finished. Then you can get fitted parameters and a curve overlaid over the raw data points. Check if the sigmoid curve is reasonably fitted over the data.
?. Set "equall to" section to "Value of 0".
This results in a warning saying "unable to find solution", but you can ignore it.
TITLE: U Mass - Basal glucose metabolism
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
Whole body glucose turnover and hepatic glucose production rates are measured at basal state using an intravenous infusion of labeled glucose in awake mice. Whole body glucose turnover and hepatic glucose production regulate basal glucose levels and are altered in obesity.</div></div>

[STEPS]
?. Survival surgery is performed to establish a chronic indwelling catheter at 5~6 days prior to experiment for intravenous infusion. (refer to M1023: Surgery-jugular vein cannulation)
?. Mice are fasted overnight (~15 hours) or for 5 hours prior to the start of experiment.
?. Place a mouse in a rat-size restrainer with its tail tape-tethered at one end.
?. Expose and flush the intravenous catheter using saline solution. Then, connect the catheter to the CMA Microdialysis infusion pump.
?. Collect plasma sample (10 µl) before the start of infusion (basal-0 min) to measure basal glucose levels.
?. Start the experiment by turning on the pump and intravenously infusing [3-³H] D-glucose at 0.05 µCi/min in awake mice. ([3-³H] D-glucose is suspended in saline solution)
?. Collect plasma samples (10 µl each) at 90, 100, 110, and 120 min to measure glucose levels.
?. Collect additional plasma samples (10 µl each) at 90, 100, 110, and 120 min to measure [3-³H] D-glucose concentrations. (10 µl plasma samples are suspended in 20 µl distilled water [dH²O] to make 30 µl sample solutions.)
?. At the end of experiment, mice are euthanized using pentobarbital.
?. Biochemical assay is conducted using plasma samples to measure [3-³H] D-glucose concentrations.
a)  Transfer 15 µl of plasma sample solutions into microcentrifuge tubes with sample time clearly labeled.
b)  Add 25 µl BaOH and vortex samples.
c)  Add 25 µl Zn(SO)² and vortex samples.
d)  Centrifuge samples for 5 min at 12,000g (~14,000 rpm).
e)  Transfer 20 µ of supernatant into scintillation vials and place into vacuum oven set at room temperature for overnight drying.
f)  Following overnight drying, add 80 µl dH²O and vortex thoroughly.
g)  Add 3 ml of Ultima scintillation cocktail and vortex sample.
h)  Measure [3-³H] D-glucose using Beckman Coulter Scintillation Counter.
?. Basal rate of whole body glucose turnover is calculated as the ratio of the [³H]glucose infusion rate to the specific activity of plasma glucose averaged for 90~120 min of experiment.
TITLE: Hornwort RNA extraction
AUTHORS: Eftychis Frangedakis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The same protocol used for DNA extraction is used for RNA extraction with the addition of an overnight RNA precipitation step with LiCl.</div></div>

[STEPS]
?. Grind 0.5-2 g of tissue using mortar and pestle in the presence of liquid nitrogen until finely ground. Transfer frozen ground tissue to a 30 ml tube.
?. Add 10 ml of 60 oC extraction buffer and 100 mg PVP-40/g tissue. Mix by inversion and incubate in 60 oC  in a water bath for 30 min (or incubate on ice for 15 min).
?. Remove from heat, and let cool to room temperature for 4 to 6 min.
?. Add 12 ml of chloroform:IAA (24:1) and mix by inversion to form an emulsion.
?. After mixing thoroughly, spin at 10,000 rpm for 10 min at RT.
?. Transfer aqueous phase to a new 30 ml centrifuge tube. Repeat chloroform:IAA extraction to remove cloudiness (PVP) in aqueous phase. Spin at 7,000 rpm for 10 min at RT.
?. Add ½ volume of 5M NaCl  to the final aqueous solution recovered. Mix well. Add two volumes of cold (-20 oC) 95% ethanol. Mix by inversion. Place in freezer (-20 oC) for 10 min to accentuate precipitation. Spin at 13,000 rpm for 6 min.
?. Optional: Resuspend in 2 ml of TE and repeat step 7.
?. Pour off supernatant. And wash pellet with cold (0 to 4 oC) 70% v/v ethanol. Dry pellet.
?. Dissolve in 400 μl TE and add 100ul 8M LiCl. Incubate o/n at 4 oC.
?. Spin 10 min to pellet RNA . Remove supernatant (contains DNA).
?. Dissolve pellet with 400ul DEPC dH2O. Add 40ul NaoAc (pH 4.5) and 1ml 95% EtOH and freeze at -80 oC for 10 min. (optional: wash pellet with 95% EtOH).
?. Spin for 10 min at 13K and resuspend pellet in 40ul DEPC dH2O.
?. If necessary (pellet not dissolved): incubate at 60 oC for 10 min and then on ice for 30 min. Pellet debris by spinning tube for 10 min at 13K. Transfer supernatant (RNA) to a new tube.Extraction buffer:100mM Tris-HCL pH 8  (2M)		25ml.