eamag/protocols-with-steps huggingface dataset:
TITLE: Biochemical Measures of Neuropathy - Glutathione Peroxidase
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. </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/biw94kcn.jpg" /></div></div>

[STEPS]
?. [Sample Preparation — Tissue:]
1. Homogenize the tissue in 5–10 mL of cold buffer (i.e., 50 mM Tris-HCl, pH 7.5, 5 mM EDTA, and 1 mM DTT) per gram of tissue.                General Equation: μL Buffer = mg Tissue X 10
2. Centrifuge at 10,000 x g for 15 minutes at 4°C.
3. Remove supernatant for assay and store on ice. Sample can be stored at –80°C for at least one month.
?. [Performing Assay:]
1. Turn on Multiskan and open file gpx.sed.2. Background Wells: add 120 μL of Assay Buffer and 50μL of co-substrate mixture to three wells.3. Positive Control Wells: add 100 μL of Assay Buffer, 50 μL of co-substrate mixture, and 20 μL of diluted GPx (control) to three wells.4. Sample Wells: add 100 μL of Assay Buffer, 50 μL of co-substrate mixture, and 20 μL of sample to three wells.5. Initiate reactions by adding 20 μL of cumen hydroperoxide to all wells being used as quickly as possible. Note precise time the reaction is initiated.6. Place plate onto Multiskan holder and click START.7. Save raw data as an Excel file into the GPx data folder. Use the naming convention gxXXXX.xls, where XXXX is the date in mmdd format.8. Select Process>Organize. Choose the appropriate data to organize (usually Measure1), then click OK. This rearranges the data into columns.9. Save organized data as an Excel file into the GPx data folder. Use the naming convention gxXXXXor.xls, where XXXX is the date in mmdd format.
TITLE: U Mass - Acute lipid infusion
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">Triglyceride emulsion and heparin will be intravenously infused for 5 hours to acutely raise circulating fatty acids levels in awake mice. Acute lipid infusion is shown to cause insulin resistance in peripheral organs.</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 fatty acids levels.
?. Start the experiment by turning on the pump and intravenously infusing 20% Intralipid (triglyceride emulsion) at 2.5 ml/kg/hr and heparin at 6 U/hr in awake mice.
?. For control experiment, 20% glycerol is infused at 2.5 ml/kg/hr in awake mice.
?. Collect plasma samples (10 µl each) at 120, 240, and 300 min to measure serum FFA levels.
?. At the end of experiment, tissues may be collected for biochemical and molecular analyses.
?. Alternatively, a 2-hr hyperinsulinemic-euglycemic clamp may follow this 5-hr acute lipid infusion experiment to measure the effects of elevated fatty acids on insulin sensitivity. (refer to M1001: Hyperinsulinemic-euglycemic clamp)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This describes setting up ssh keys and configuration to avoid having to use your NetID+ 2-factor authentication every time you log into the HPC.  Windows users are encouraged to install Cygwin (http://www.cygwin.com), a free Unix-like environment that provides a terminal so as to avoid using an ssh client like Putty.  Cygwin users must be sure to install the "ssh" tools which are not installed by default.</p>

[STEPS]
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TITLE: Protocol_Impact of a telephone triage service for non-critical emergencies in Switzerland: a cross-sectional study
AUTHORS: Chloé Thierrin, Aurélie Augsburger, Fabrice Dami, Christophe Monney, Philippe Staeger, Carole Clair

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left"><span>BACKGROUND:</span><span style = "font-weight:bold;"> </span><span>Telephone triage services (TTS) play an increasing role in the delivery of healthcare. The objective of this study was to characterize the users of a TTS for non-critical emergencies, describe the types of advice given and their subsequent observation, and assess the influence of TTS on the use of the healthcare system in a sanitary region of Switzerland.         </span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">METHOD: Data from a TTS based in the French part of Switzerland will be analyzed. This service consists of a medical contact center for non-critical emergencies, with trained nurses available 24/7. We created a questionnaire based on literature search and inspired by other questionnaire to assess these different points. All calls for medical complaints from adults (people aged > 18 years old) during a 3 months period (from July 2018 to September 2018) will be listed. 20 calls per day will be randomly selected with the software STATA. Research students will contact users 2 to 4 weeks after the initial call to reach a total sample of 430 users. In this descriptive study, the main outcome is to evaluate if the call to TTS change the attitude of care (dichotmomous variable yes/no). We expected a change in 20% of users of CTMG (p = 0.2) with a 99% confidence interval of (15% - 25%). Using the general formula ?=??/22?(1−?)/?2 we find n = 425, being e=0.05 the half width of the desired confidence interval, ?=0.01 the 1’s complement of the confidence level (99%), and ??/2 the (1−?/2) quantile of the standard normal distribution (?=0.01 → ??/2 =2.58). We will use STATA15.0 to perform the statistical analyses.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. [Time table]
Study materials
?. [Time table]
Study materials
?. [Time table]
Ethical review
?. [Time table]
Data collection
?. [Time table]
Coding/Analyzing data
?. [Time table]
Writing the manuscript
?. [Time table]
Ethical review
?. [Time table]
Data collection
?. [Time table]
Coding/Analyzing data
?. [Time table]
Writing of the manuscipts
TITLE: 4% Formaldehyde in .1M PB (FOR IMMUNO BRAINS) (1L)
AUTHORS: Molly Brennan, Olivier George

[STEPS]
?. .4M Phosphate buffer pH 7.4 (250mls)21.7 g Na2 HPO4 (sodium phosphate dibasic-heptahydrous)                                [86.8 g]~Make sure dibasic completely dissolves before adding monobasic!        2.6 g Na H2 PO4 (sodium phosphate monobasic-monohydrous)                            [10.4 g]Dissolve in 200 ml of distilled H2O                                                                                [800 mls]pH to 7.4Bring to 250ml volume with distilled H2O
?. In separate 1000 ml volumetric flask:[2000ml]2.250 ml 0.4 M phosphate buffer (pH=7.4)                                                                        [500ml]3.100 ml 37% formaldehyde                                                                                                   [200mls]4.Bring to 1L volume with distilled water (no need to re-pH)                                                2L]5.Filter using side-arm flask (use glassware labeled ‘paraform’)**Please use gloves, protective eyeware and mask when working w/ formaldehyde.Label and date 1L 4% Formaldehyde and put in plastic bottle marked“4% FORM IMMUNO”.Put in black KOOB fridge (by -80 freezer).
?. 0.15 M Saline Pre-fixative Rinse (1 L)                                                    [2L]1.Add 9.0 g NaCl to 800 mL dH2O.                                                                    [18g]2.Add ~1000 units of heparin per 1L saline                                                    [2mls]3. Stir until dissolved (good for 1 week).4. AdddH2Oto bring to volume.5. Label and refrigerate
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Human Islet Purification - COBE setup
AUTHORS: James Lyon, Patrick MacDonald, Jocelyn Manning Fox

[STEPS]
?. [Purge]
Turn on COBE 2991 processors and purge as follows:Set dials; centrifuge speed to 3000 rpm, super out rate to 450 mL/min and super out volume to 600 mL.Install foam ring and plastic shim in to COBE.Press START/SPIN, when rpm are at 3000, press SUPER OUT.Once alarm sounds press STOP/RESET.Allow COBE 2991 bowl to drain before placing cell processing bag in processor.Set dials; centrifuge speed to 1500 rpm, and super out rate to 0 mL/min.
?. [install gradient maker]
Open sterilized gradient maker set and place sterilized stir bar in front beaker of gradient makerPlace assembled gradient maker set on top of magnetic stir plate.Clamp tubing between beakers with haemostats.Place section of the COBE connector tubing through Masterflex pump with open end of tubing facing adjacent COBE 2991, and close latch.
?. [Install COBE tubing/bag set]
Open centrifuge latch by rotating clockwise, remove metal seal weight, and open sliding covers.Remove locking cover by rotating counter clockwise while holding up locking pin.Remove white alignment blocks, small plastic cover, and foam insert.Feed sterile cell processing bag through centre hole of locking cover.Install bag in centrifuge bowl by aligning holes in bag over studs on bowl. Spread bag out evenly in attempt to eliminate creases.Place white alignment blocks with bevelled end facing up around center stem of bag. Place locking cover around blocks and over studs. Rotate cover clockwise until locking pin drops into place.Close sliding covers, place metal seal weight on hexagonal seal of cell processing bag and close centrifuge latch by rotating counter clockwise.Clamp all bag lines near manifold with haemostats, except for top left or right line depending on pump orientation.Support main bag line upright by placing all clamped lines through pinch valves on COBE 2991Place end of main line in Biosafety Cabinet, remove line cap and connect to end of COBE connector tubing.
?. [Prepare tissue collection conical tubes]
Open two packages of sterile 250 mL conical tubes and place into 250 mL racks.Label 250 mL conicals as numbers 1 to 12. If there is more than 1 purification run the conical tubes shall also be designated with a letter (E.g. A1 - A12, B1 - B12…).Refer to Human Islet Isolation Media Preparation protocol.Fill conical 1 with 100 mL of wash 2 solutionFill conical 2 with 150 mL of wash 2 solution.Fill conicals 3 – 4 with 200 mL of wash 2 solution.Fill conicals 5 – 11 with 225 mL of wash 2 solution.Fill conical 12 with 100 mL of dilution 1 solution.Place conicals in refrigerator until use.
TITLE: Change Release Protocol
AUTHORS: Abhishek Srivastava

[DESCRIPTION]
This is a set of protocols for releasing the changes or new feature to staging and production. This in being introduced so as the developer does not skip any critical step required during the whole release process. Any critical step if is missed could lead to incidents in production environment.

[STEPS]
SECTION: Merge checklist Staging
1. Merge checklist Staging
SECTION: Merge checklist Staging
1.1. Regen pb.go from shared-proto master branch
SECTION: Merge checklist Staging
1.5. Test in local
SECTION: Merge checklist Staging
1.6. Test in feature env: <name of the feature environment>
SECTION: Merge checklist Staging
1.7. Test Party
SECTION: Merge checklist Staging
1.4. Run Migration Scripts if any on Staging
SECTION: Merge checklist Staging
1.2. Create Gitlab PR for config change on Staging
SECTION: Merge checklist Staging
1.3. Merge Gitlab PR for Staging
SECTION: Merge checklist Production
2. Merge checklist Production
SECTION: Merge checklist Production
2.4. Merge shared-proto change (if applicable)
SECTION: Merge checklist Production
2.1. Run Migration Scripts if any on Production
SECTION: Merge checklist Production
2.2. Create Gitlab PR for config change on Staging
SECTION: Merge checklist Production
2.3. Merge Gitlab PR for Staging
SECTION: Starting the Jenkins Pipeline
3. Starting the Jenkins Pipeline. Cutoff 3pm unless absolutely necessary/hot-fix
SECTION: Starting the Jenkins Pipeline
3.1. Announce on #backend-release channel
SECTION: Starting the Jenkins Pipeline
3.2. Check the result of QA test suite run on #qa-backend-releases slack channel
 

SECTION: Starting the Jenkins Pipeline
3.3. If any failures then check with the QA engineers
SECTION: Starting the Jenkins Pipeline
3.4. Get a go ahead from QA engineers to proceed in case of failures
SECTION: Starting the Jenkins Pipeline
3.5. Make sure config changes done
SECTION: Starting the Jenkins Pipeline
3.6. Make sure migration done if required
SECTION: Starting the Jenkins Pipeline
3.7. Proceed to Canary
SECTION: Validate Changes on Canary
4. Validate Changes on Canary
SECTION: Validate Changes on Canary
4.1. Check logs on Canary Server
 

SECTION: Validate Changes on Canary
4.2. Check for errors related to your grpc method


 

SECTION: Validate Changes on Canary
4.3. Verify Changes on Canary if possible using port-forwarding
SECTION: Validate Changes on Canary
4.4. Check Grafana Dashboards for Hystrix and GRPC for error rates on New Canary pod


SECTION: Validate Changes on Canary
4.5. Wait for another 1 hour at least for baking the changes in Canary [Mandatory]  

SECTION: Validate Changes on Canary
4.6. Check Grafana Dashboards for Hystrix and GRPC for error rates on New Canary Pod
SECTION: Validate Changes on Canary
4.7. If all good, proceed for all server roll-out
SECTION: Validate Changes on Canary
4.8. Verify Changes by completing the whole new flow on app/web to see all good with new feature/ old feature
SECTION: Validate Changes on Canary
4.9. Attach Screenshots from test on production
SECTION: Validate Changes on Canary
4.10. Update the PR Template
TITLE: Wastewater grab sample processing with PEG-8000 precipitation 
AUTHORS: Dilip Abraham, Nirmal kumar, Vinoth kumar, Ganesh Rajamanickam, Venkata Raghava Mohan

[DESCRIPTION]
PEG (Polyethylene Glycol) is a chemically inert, nontoxic, water-soluble synthetic polymer and has been used in aqueous polymer two-phase systems that help concentrating and isolating viruses from a variety of environmental samples. PEG is known as a good inductor of attractive interactions that crystallize viruses in the interpolymer spaces between PEG molecules. So, the combination of PEG concentration and RNA extraction steps enable 900– 1500X concentration of wastewater samples and sufficiently eliminate most of the organic matter, which could inhibit the subsequent qPCR assay.

[STEPS]
SECTION: Sample
1. Wastewater grab samples

SECTION: Procedural steps - PEG 8000 concentration method:
2. Collect  350 mL   of sewage samples in a sterile Whirl-Pak bag and disinfect the sampling bag with ethanol to avoid contamination.
SECTION: Procedural steps - PEG 8000 concentration method:
3.  Transfer the bag to the laboratory in a cold chain 4 °C .
SECTION: Procedural steps - PEG 8000 concentration method:
4. The sewage sample is transferred to a sterile 250 mL Nalgene centrifuge bottle.
SECTION: Procedural steps - PEG 8000 concentration method:
5. Add 25 g  of PEG 8000 and  5.6 g of NaCl in 250 mL  of  the sample to get the final concentrations of   (w/v) and  0.3 Molarity (M)  respectively.
SECTION: Procedural steps - PEG 8000 concentration method:
6. Vortex the mixture at   for 15 min  until the reagents are completely dissolved in the suspension.
SECTION: Procedural steps - PEG 8000 concentration method:
7.  Thereafter, seal the Nalgene bottles using the cap assembly, and centrifuge the mixture at 12000 x g, 120 min until the pellet is visibly seen.
SECTION: Procedural steps - PEG 8000 concentration method:
8.  Resuspend the pellet in 3 mL  of PBS (pH 7.4). Then, aliquot 1 mL  of the suspension into each of 3 screw cap tubes.

SECTION: Procedural steps - PEG 8000 concentration method:
9. The resulting pellet is stored at -20 °C  until further process.

Freeze-thawing of primary concentrates is limited to once.
TITLE: Growing overnight culture of OP50 as worm food
AUTHORS: Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Luria broth (LB) is a nutrient-rich media commonly used to culture bacteria in the lab. LB agar plates are frequently used to isolate individual (clonal) colonies of bacteria carrying a specific plasmid. However, a liquid culture is capable of supporting a higher density of bacteria and is used to grow up sufficient numbers of bacteria necessary to isolate enough plasmid DNA for experimental use. The following protocol is for inoculating an overnight culture of liquid LB with bacteria. </div><div class = "text-block">This protocol is specifically for making a liquid bacterial culture of OP50 and the following parameters are specific for this bacterial strain:</div><div class = "text-block">1. Name of the bacterial strain: E.coli (OP50)</div><div class = "text-block">2. Growth temperature: 37C</div><div class = "text-block">3. Incubation time: 16-18hrs (Overnight)</div><div class = "text-block">4. Rpm of the shaking incubator: 200-220 rpm</div></div>

[STEPS]
?. Obtain LB Broth from the Media kitchen- Make sure to make at least 500ml in volume for standardisation purposesLB Broth contents: 4gNaCl4 g Tryptone2 g Yeast Extract dH2O to 400 mL
?. Obtain sterile Erlenmeyer flask from the media kitchen
?. Wipe the work area with 70% ethanol and create a relatively sterile environment on the laboratory bench by using a simple gas-powered burner
?. Add the desired volume of liquid LB to the flask
?. Using a sterile inoculation loop, select a single colony from your bacteria streaked LB agar plate (One streaked plate can be used for about a month and stored at 4C during that time period)
?. Dip the inoculation loop into the liquid LB and swirl. Discard the inoculation loop
?. Loosely cover the culture with sterile aluminium foil or a cap that is not air tight as bacteria needs air
?. Incubate the bacterial culture at the required growth temperature overnight in a shaking incubator
?. After incubation, check for growth, which is characterized by a cloudy haze in the media
?. Measure the optical density of the bacterial culture at 600nm wavelength using a spectrophotometer. Record the OD600 three times and calculate average, use LB Broth as Blank
TITLE: Polymerase chain reaction (PCR)
AUTHORS: Shuning   Guo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to amplify target DNA fragment for plasmid construction or other use.</div></div>

[STEPS]
?. Choose one case from the cases below.
?. Add the following reagent to a PCR tube.(50 μl).2×High Fidelity Master Mix (MCLAB)25 μlTemplate1 μlForward Primer (10 μM)1 μlReverse Primer (10 μM)1 μlddH2O22 μl
2×High Fidelity Master Mix (MCLAB)25 μlTemplate1 μlForward Primer (10 μM)1 μlReverse Primer (10 μM)1 μlddH2O22 μl
?. Program the thermocycler as follows:TemperatureTime95/98℃5 min95/98℃30 sTm-3~5℃30 s72℃1kb/min72℃5~10 min16℃∞
TemperatureTime95/98℃5 min95/98℃30 sTm-3~5℃30 s72℃1kb/min72℃5~10 min16℃∞
?. Use the palm centrifuge to mix the solution in PCR tube.
?. Put the PCR tube into the thermocycler and Run the program.
?. Using agarose gel electrophoresis to confirm if correct construct was present.
?. Pick colonies as the template for colony PCR. Mix the colonies with 2.5μl ddH2O and pick 1μl as PCR template and 1.5μl for culture.
?. Add the following reagent to a PCR tube.(10 μl).2×Raqid Master Mix (Vazyme)5 μlTemplate0.4 μlForward Primer (10 μM)0.4 μlReverse Primer (10 μM)0.4 μlddH2O3.8 μl
There is no need to add Gold View as colouring agent for agarose gel eletrophoresis when using 2×Rapid Master Mix (Vazyme) as PCR enzyme.
2×Raqid Master Mix (Vazyme)5 μlTemplate0.4 μlForward Primer (10 μM)0.4 μlReverse Primer (10 μM)0.4 μlddH2O3.8 μl
?. Program the thermocycler as follows:TemperatureTime95/98℃5 min95/98℃30 sTm-3~5℃30 s72℃2kb/min72℃5~10 min16℃∞
TemperatureTime95/98℃5 min95/98℃30 sTm-3~5℃30 s72℃2kb/min72℃5~10 min16℃∞
?. Use the palm centrifuge to mix the solution in PCR tube.
?. Put the PCR tube into the thermocycler and Run the program.
?. Using agarose gel electrophoresis to confirm if correct construct was present.
TITLE: Downloading Viral Metagenome Data
AUTHORS: Benjamin Bolduc

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Downloading reads from the </span><a href="https://github.com/MicroB3-IS/osd-analysis" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Ocean Sampling Day (2014)</span></a><span> using fastq-dump, a tool from the SRA toolkit implemented in </span><a href="cyverse.org" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Cyverse</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Download from SRA]
Open FASTQ-dump
?. [Download from SRA]
Specify the SRA accession
?. [Getting Started]
This is the very first step in downloading reads from Ocean Sampling Day (2014). If you navigate through their wiki and other documentation, you can find the SRA associated with this dataset: ERR771003.There are a lot of ways to download this data and get it on CyVerse. The following are a few ways:Download the data from SRA manually and then use Cyberduck, follow CyVerse's Cyberduck instructions.Download the data from SRA manually and then upload via iCommands.For this, we'll use the FASTQ-dump tool available from the SRA, which is also available on CyVerse.
?. [Download from SRA]
Launch AnalysisRun the job!
?. [Results]
After the job finishes, you'll have a directory with two important files, ERR771003_1.fastq and ERR771003_2.fastq.You can use these files directly in the next step, or compress them if you want to save disk space.
TITLE: CDPH modified Artic v3 primers for SARS-CoV-2 sequencing
AUTHORS: John Bell

[DESCRIPTION]
This protocol lists the primer bed file for a modified Artic v3 set for SARS-CoV-2 sequencing.   Two primers have been added to reduce dropouts.

[STEPS]
1. The attached bed file is a modified Artic v3 primer location list for SARS-CoV-2, used for sequencing by California Dept. of Public Health Viral and Rickettsial Disease Laboratory during the COVID-19 pandemic.