eamag/protocols-with-steps huggingface dataset:
TITLE: Pre-Imaging Liquid Growth Medium - Yeast
AUTHORS: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald

[DESCRIPTION]
This protocol describes the steps to prepare liquid culture medium for Saccharomyces cerevisiae. This liquid medium is used to optimize yeast cultures for fluorescence imaging by the reduction of auto-fluorescence through an abundance of Adenine [1].

[BEFORE_START]
Have the following solutions premixed:

Glucose 20% 500 ml solution:
Concentration: 200 g/l
mix 100 g Glucose in 500 ml deionized water (ddH2O)

Adenine 100x 100 ml solution:
Concentration 3 g/l
mix 0.3 g Adenine in 100 ml ddH2O

Optional:
SC-xx 10x 100ml solution:
Concentration: 19.2 g/l
mix 1.92 g into 100 ml ddH2O

YNB 20x 100ml solution:
Concentration: 134.4 g/l
mix 13.44 g into 100 ml ddH2O

[STEPS]
SECTION: Preparation of 500ml liquid pre-imaging growth medium
1. Compound medium for autoclave
SECTION: Preparation of 500ml liquid pre-imaging growth medium
1.1. Fill a 500 ml flask with 445 mLddH2O. 
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 500ml liquid pre-imaging growth medium
1.2. Add 3.36 g Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids (YNB)
SECTION: Preparation of 500ml liquid pre-imaging growth medium
1.3. Add 0.86 g SC-XX powder. 
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium
1.4. Add 5 mLAdenine 100x solution.
 


SECTION: Preparation of 500ml liquid pre-imaging growth medium
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium
3. When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
SECTION: Preparation of 500ml liquid pre-imaging growth medium
4. 
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium
1.1. Fill a 500 ml flask with 320 mLddH2O. 
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 500ml liquid pre-imaging growth medium
1.2. Add 25 mLYNB 20x solution (Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids).
SECTION: Preparation of 500ml liquid pre-imaging growth medium
1.3. Add 100 mL SC-XX 10x solution.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium
1.4. Add 5 mLAdenine 100x solution.
 


SECTION: Preparation of 500ml liquid pre-imaging growth medium
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 500ml liquid pre-imaging growth medium
3. When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
SECTION: Preparation of 500ml liquid pre-imaging growth medium
4.
TITLE: EVALUATION OF TEST ITEMS EFFICACY IN PROMOTING STROKE REHABILITATION IN THE RAT STROKE MODEL (t-MCAO).
AUTHORS:  ,  

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The objective of this protocol is to present the details of the procedures for evaluating test items’ (such as small molecules, biologics, and others) efficacy in promoting stroke rehabilitation, in the rat transient Middle Cerebral Carotid Occlusion (t-MCAO) model.</div></div>

[STEPS]
TITLE: Mask-Based Covid-10 testing system using Exhaled Breath Condensate
AUTHORS: John Daniels

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The Kinaptic mask-based COVID-19 testing system uses a unique Exhaled Breath Condensate (EBC) collector that converts breath vapor to a liquid biosample for rapid testing and wireless automatic contact tracing. Exhaled Breath Condensate (EBC) is collected from a mask-based EBC collector as a low cost disposable component. Our electronic biosensor detects even low concentrations of virus biomarkers if they are present in the EBC sample. Corona viruses have slowly evolving N-protein and quickly evolving S-protein. N-Protein testing screens for </span><span style = "font-style:italic;">any</span><span> corona virus infection / S-Protein screens for </span><span style = "font-style:italic;">only</span><span> COVID-19 infection. The Kinaptic mask-based COVID-19 testing system is a “two-stage filter”: 1) catch only of N-protein biomarker = trigger warning to get more stringent diagnostic and contact trace; 2) catch both N- and S-, or just S- biomarkers = more urgent need for immediate quarantine and medical attention. As the target biomarkers bind to the capture molecules, the electrical conductivity of the electronic biosensor changes. This change in conductive is amplified by our bluetooth electronics, analyzed to determine the presence and quantity of the target biomarker, and the test result wireless transmitted to a smartphone or directly to the cloud.</span></div></div>

[STEPS]
?. Remove release liner from double stick tape on electronics
?. Remove release liner from double sided tape on mask
?. Pull tab to Activate Battery and Adhere electonics to mask
?. Put mask on and breath normally.
?. Open APP on Smartphone
?. Connect to Mask Electronics
?. Continue to breath normaly
?. Testing is Complete
?. Upload results
TITLE: U Mass - Hyperglycemic clamp
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Hyperglycemic clamp measures insulin secretion and pancreatic -cell function in awake mice. Islet function is affected by obesity and insulin resistance, and abnormal islet function predisposes to the development of type 2 diabetes.</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 (20 µl) before the start of infusion (basal-0 min) to measure basal glucose and insulin levels.
?. Start infusion of 20% dextrose to quickly reach a target hyperglycemia (~300 mg/dl glucose level) and maintain hyperglycemia by adjusting glucose infusion rates.
?. Collect plasma samples (10 µl each) at 10, 20, 30, 45, 60, 90, and 120 min to measure glucose levels. Adjust glucose infusion rates based on instantaneous glucose levels to maintain at target hyperglycemia.
?. Collect additional plasma samples (10 µl each) at 10, 20, 30, 45, 60, 90, and 120 min to measure insulin concentrations.
?. At the end of experiment, mice are euthanized, and pancreas may be collected for further studies.
?. For data analysis, plasma insulin concentrations may be plotted during the 120-min hyperglycemic clamp experiment, and area-under-curve may be calculated. Area-undercurve of insulin levels during hyperglycemic clamps may be directly correlated with insulin secretion and pancreatic -cell function assuming there are no effects on insulin clearance rates.
?. Additional plasma samples may be collected to measure serum c-peptide concentrations which may further reflect glucose-induced insulin secretion and pancreatic -cell function in awake mice.
TITLE: Initial Installation of the Uavionix PingRID 
AUTHORS: marshall.bennett

[DESCRIPTION]
Instructions for the initial installation and registration of the Uavionix PingRID device. 

[STEPS]
1. Unbox the PingRID unit. Save the cardboard tag from the packaging with the PingRID serial number on it. 
2. Record the PingRID, SSID, and Password information from the device (included in the packaged materials) with the drone's stored records and registration information. 
3. Charge the PingRID unit using the provided USB-C cable. Test the unit to ensure that it will charge, turn on, and operate. The RID unit's orange charging light will illuminate when the device is charging and will extinguish when the device is fully charged. The device's green GPS lock light will flash slowly if it is acquiring GPS signal, remain solid green if a GPS lock has been acquired, give 2 rapid flashes if a takeoff is detected, and give 4 rapid flashes if an error occurs. 
4. Log into the FAA's Drone Zone website (https://faadronezone-operator.faa.gov/) 



5. Launch the Drown Owner and Operator's Dashboard. 
6. Click "Manage Device Inventory" 
7. Select the drone that you want to attach to the RID device to from the list of drone's that are registered to you. Click the "three dot" menu button on the right side of the dashboard's menu and select the EDIT" option to access the device's FAA Registration Records. 
8. In the "Does your drone have remote ID?" tab, select yes. 
9. Enter the remote ID serial number from the device's packaging and select the UAS type as "Remote ID Broadcast Module" in the menu that opens. Save and exit this menu. 
10. Review the drone registration information in the Device Inventory. The records should reflect that a remote ID device is provided for this drone. 
11. Attach the RID to the drone using the provided velcro and store the drone and RID device until ready to be used. 
12. The PingRID device is ready to use. When you're ready to fly, follow the Uavionix Quick Start Guide from the Uavionix website.
TITLE: Immunopeptidomic analyses of RAW 264.7 macrophages
AUTHORS: Joel Lanoix, Pierre Thibault

[DESCRIPTION]
Here, we describe protocols for a novel proteogenomic approach to identify MHC I–associated peptides from RAW 264.7 cells after LPS stimulation. These antigens are rare and are most effectively identified with a mass spectrometry–based approach, which allows the direct sampling and sequencing of these peptides.

[STEPS]
TITLE: Investigation of the pain factors of rotator cuff tears: a protocol for a scoping review
AUTHORS: Tomohisa Yuda, Katsuya Sakai, Takashi Saito, Hayato Shigetoh, Masateru Hayashi, Takashi Kitagawa

[DESCRIPTION]
Objective：
The purpose of this scoping review is to investigate factors associated with rotator cuff tear pain and summarize them into incidence, exacerbation, and remission factors.
 
Introduction：
Rotator cuff tears are one of the most common shoulder disorders. A variety of associated factors have been cited in observational studies, and systematic reviews have examined factors associated with symptomatic rotator cuff tears. However, no reports differentiate pain factors into developmental, exacerbating, and remitting factors.
 
Inclusion/exclusion criteria：
The study will include patients whose physicians have diagnosed non-traumatic rotator cuff tears using Magnetic Resonance Imaging or ultrasound echo. Outcomes will be a pain, the severity of rotator cuff tear, physical function, and psychological factors. The study design will be observational, with no restrictions on region, race, gender, or language of the original paper.
 
Methods：
A systematic search of PubMed, Web of Science, CINAHL(EBSCO), PEDro, and Embase (Dialog) databases using the keywords “rotator cuff tear,” “pain,” and “etiology” will be conducted during March 2024. In the first screening step, two independent reviewers will review all of the titles and abstracts to exclude irrelevant articles. In the second screening step, two independent reviewers will review all of the full texts to exclude irrelevant articles. Outcomes will focus on rotator cuff tear severity, pain, physical function, and psychosocial factors, categorizing factors associated with pain according to each study design and identifying incidence factors, exacerbating factors and remission factors.

[STEPS]
TITLE: Recan: R-based tool for detection of recombination in viral genomes
AUTHORS: Vasily Pavelko

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Recan is an R version of the Python library that allows the identification of recombination events through construction and exploration of similarity plots based on genetic distances between nucleotide sequences. After porting I validated this package using sequences of viruses with previously identified recombitations. During benchmarking both versions demonstrated the same execution time. </div><div class = "text-block"><span>To benchmark the packages we selected the same four sets of viral genomes used in the Python version of recan </span><a href="https://www.zotero.org/google-docs/?broken=1VahDR" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">(Babin, 2020)</span></a><span>. </span></div></div>

[STEPS]
?. [Benchmarking of recan package in R]
Calculating average execution time of code using Hepatitis C virus datasets.
?. [Benchmarking of seqcombo package in R]
Calculating average execution time of code using Lumpy Skin Disease Virus datasets.
?. [Benchmarking of recan package in R]
Calculating average execution time of code using Human Immunodeficiency Virus datasets.
?. [Benchmarking of recan package in R]
Calculating average execution time of code using Norovirus datasets.
?. [Benchmarking of recan package in R]
Calculating average execution time of code using Lumpy Skin Disease Virus datasets.
?. [Benchmarking of seqcombo package in R]
Calculating average execution time of code using Human Immunodeficiency Virus datasets.
?. [Benchmarking of seqcombo package in R]
Calculating average execution time of code using Hepatitis C virus datasets.
?. [Benchmarking of seqcombo package in R]
Calculating average execution time of code using Norovirus datasets.
?. [Benchmarking of recan library in Python]
Calculating average execution time of code using Hepatitis C virus datasets.
?. [Benchmarking of recan library in Python]
Calculating average execution time of code using Human Immunodeficiency Virus datasets.  Each step I run 2 times as a preparatory steps, and then 7 runs used for calculation average time.
?. [Benchmarking of recan library in Python]
Calculating average execution time of code using Norovirus datasets.
?. [Benchmarking of recan library in Python]
Calculating average execution time of code using Lumpy Skin Disease Virus datasets.
TITLE: Cryopreservation of marine diatoms
AUTHORS: Jana Hinners, Sinead Collins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Cryopreservation and thawing protocol for </span><span style = "font-style:italic;">Thallasiosira </span><span>diatoms.</span></div></div>

[STEPS]
?. [Prepare diatom cultures]
Grow 40mL diatom cultures in 50mL flasks until mid to end exponential phase.Do not invert samples for at least 24 hours before cryopreservation, so that diatoms are accumulated close to the bottom of the flask
?. [1 day before cryopreservation]
Prepare fresh culture media (e.g. f/2) with 24% DMSO (=cryoprotective agent)Depending on how dense the cultures are, the concentration of DMSO can be altered.The final concentration of DMSO in the cryopreserved sample should be 12%.
?. [Cryopreservation day]
Concentrate the phytoplankton samples by removing the upper 30mL of each culture flask without disturbing the diatom biomass accumulated at the bottom
?. [1 day before cryopreservation]
Cool down Isopropyl alcohol (IPA) to 4 degrees Celsius
?. [Cryopreservation day]
Keep light intensity low for this step.Mix concentrated phytoplankton sample 50:50 with the DMSO-media to obtain a 12% DMSO-concentration in the sample.
?. [Cryopreservation day]
Keep light intensity low for this step.Incubate samples for 15-30 min with DMSO-media.In this time transfer replicates to cryovials, each receiving a volume of 1.6-1.8 mL depending on the chosen cryovial type.Aim for at least 6 replicates per sample, since the survival rate of cryopreserved samples can vary.I find it useful to handle 18 replicates at a time because they fit into 1 Mr Frosty.Store the cryovials in the chilled Mr Frosty.
?. [Cryopreservation day]
Fill Mr. Frosty with the 4 degree cold IPA.
?. [Cryopreservation day]
Store Mr. Frosty with cryovials at -80 degrees Celsius for at least 3-5 hours.Do not close the Mr. Frosty too strong, otherwise it is hard to open the lid again.
?. [Cryopreservation day]
After the 3-6 hours, remove the frozen cryovials from the Mr. Frosty, place them in a clearly labelled Cryobox (make sure to use a cryopservervation-safe label). Make sure you store the cryovials in a clear order and note down the order for long-term accessibility.Store the cryobox in liquid nitrogen and note down the exact location of the box for long-term accessibility.Success of the cryopreservation can be controlled after at least 24h in liquid nitrogen.
?. [Thawing procedure]
Keep light intensity low for this step.Thaw cryovials in a 37 degrees Celsius water bath until all water crystals disappeared (ca. 2 min).
?. [Thawing procedure]
Keep light intensity low for this step.Dilute samples into 45 mL fresh media to dilute the DMSO.
?. [Thawing procedure]
Keep light intensity low for this step.Place diluted sample into 4 degrees Celsius in the dark for 12 hours.
?. [Thawing procedure]
After 12h increase the temperature to standard growth temperature.After another 12h increase the light intensity to standard light intensity.
?. [Thawing procedure]
Recovery and growth of cultures may take several days to weeks.
TITLE: Wire Hang Test 
AUTHORS: Sabina Marciano, Tae-In Kam, Roberta Marongiu, Ted Dawson

[DESCRIPTION]
This behavioral test is used to assess motor deficits in mouse models of Parkinson's disease.

[STEPS]
2. Hold the mouse by the tail and place it gently so that only its forepaws reaches and holds the wire. As soon as the animal is properly suspended, start the timer. After being released, most animals catch the wire with the four limbs. This is allowed.
3. If the mouse reaches one of the ring stands, stop the timer.
Record the time. The “reaching” score increases by 1.
4. Replace the mouse on the center of the wire and continue testing.
5. If the mouse falls, the timer is stopped. The time is recorded and its ”fall” score is decreased by 1.
7. The test ends when the timer reaches 180s or if the fall score reaches 0, i.e. the mouse has fallen 10 times. 
1. Set up the wire across the two ring stands according to the measurements listed in the materials section. Ensure the wire is tightly bound.

Note that for the protocol listed below, the "reaching" score starts at 0, while the "fall score" starts at 10. 
8. Analysis: The final reach and fall scores and measured for all animals tested. A survival curve can also be created using the timestamps for each instance of either the fall or reach score changing.
6. Replace the mouse on the center of the wire and continue testing.
TITLE: Preparation of 5M NaOH
AUTHORS: Nicola O'Reilly

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purpose of examination / Clinical relevance</span></div><div class = "text-block">At the end of 2019, several pneumonia cases were reported in Wuhan, China and the pathogen was confirmed as a new viral strain. World Health organization has named the newly identified coronavirus as 2019-nCoV, also known as COVID19. The disease developed into a dangerous pandemic, posing major challenges to the NHS. Although more research is necessary to better understand the virus, in response to the emergency, simple and rapid testing is essential to identify the virus in infected individuals. This will aid the implementation of efficient interventions to contain the spread, and distinguish healthcare workers who have been infected, and are required to self-isolate, from those showing similar symptoms but which are not 2019-nCoV associated. The latter category may continue to work, alleviating stress on hard-pressed healthcare resources. 2019-nCoV is an RNA virus, and the diagnostic tests detect viral RNA in swabs from patient airways using a reverse transcriptase PCR assay. Samples are submitted to HSL, an accredited reporting laboratory, and transferred to the Crick for testing.</div><div class = "text-block"><span style = "font-weight:bold;">Principles of Examination</span></div><div class = "text-block"><span>This procedure involves the preparation of 5M NaOH for making </span><a href="https://www.protocols.io/private/D1949D20846811EA9B0C0242AC110002" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">L6 virus inactivation buffer</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Preparation of 300 ml 5M NaOH]
Weigh out . Add to 500 mL beaker.
Work in fume hood!
[NaOH]
?. [Preparation of 300 ml 5M NaOH]
Measure out  and add to beaker.
[milliQ water]
?. [Preparation of 300 ml 5M NaOH]
Transfer to a 500 ml graduated cylinder. Make up to 300 ml using milliQ water, if necessary.
?. [Preparation of 300 ml 5M NaOH]
Label with batch and date.
?. [Preparation of 300 ml 5M NaOH]
Store solution at  ( – ).
0 Room temperature
15 °C
25 °C
?. [Preparation of 300 ml 5M NaOH]
Transfer to a 500 ml Duran bottle.
TITLE: Haematoxylin-Eosin stain for cryosections
AUTHORS: Simon Blanchoud

[STEPS]
?. [Groat's haematoxylin]
Mix:	-   of  Ammonium iron(III) sulfate [Iron alum] (CAS 7783-83-7)	-  of dH2O	-  of sulfuric acid (CAS 7664-93-9)
1 g
50 mL
0.8 g
?. [Groat's haematoxylin]
Mix separately:	-   of hematoxylin (CAS 517-28-2) dissolved in  of EtOH (96%)
0.5 g
50 mL
?. [Groat's haematoxylin]
Pour the water-based mix into the EtOH one, filter if necessary.
?. [0.1% HCl in 70% EtOH]
Mix:	-  of HCl ( )	-   of EtOH ( )Solution can be stored and used to differentiate ~30 slides.
250 µl
100 mL
?. [Eosin Y 0.1%]
Mix:	-   of Eosin Y (CAS 17372-87-1)	-  of dH2O	-  of glacial acetic acidFilter. Erythrosine B (CAS 16423-68-0) could be used instead of Eosin for a redder coloring.
0.1 g
100 mL
0.5 mL
?. [Histo-Clear II (HC)]
From National Diagnostics (HS-202). A safer alternative to Xylol for the final washes.Requires mounting with Omnimount from National Diagnostics (HS-110).
?. [Staining protocol]
?. [Staining protocol]
Thaw and rehydrate the cryosection in tap water
?. [Staining protocol]
Stain in Haematoxylin
?. [Staining protocol]
Differentiate each slide by shaking it vigorously in HCl-EtOH
?. [Staining protocol]
Place in a coplin jar with tap water, place the jar in the sink and let tap water run over it for
?. [Staining protocol]
Rinse in distilled water.
?. [Staining protocol]
Stain in Eosin Y for
?. [Staining protocol]
Rinse in EtOH  (slides could be checked for their Eosin staining).
?. [Staining protocol]
Start the wash “baths”: EtOH (80%, 100%, 100 %) & HC (I, II, III)	 each.
?. [Staining protocol]
Mount using Omnimount