eamag/protocols-with-steps huggingface dataset:
TITLE: Performance and Safety of Vaginal Administration of Tocopherol Acetate (Vitamin E) in Pre-, Peri-, and Post-Menopausal Women
AUTHORS: Prof. Francesco Cosentino, Prof. Roberto Di Marco

[DESCRIPTION]
The vaginal microbiota has a significant bearing on a woman's mental and physical health at every stage of life. A balanced microflora of the vaginal environment (eubiosis) consists of a predominant presence of lactic acid bacteria. Lactobacilli ensure the maintenance of an acidic pH that effectively makes the environment inhospitable to pathogens by preventing infection and protecting the genital tract. At the end of the reproductive period, the reduction of sex hormones, particularly estrogen, results in a decrease in the lactobacillus component with consequent depletion of the vaginal microbiota and increased susceptibility to pathogens.
Tocopherol (vitamin E) is an essential human nutrient with high antioxidant power. Topically administered, it not only plays a key role in protecting epithelial cell membranes from oxidative damage but is also able to modulate bacterial growth by contributing to pH acidification. The latter feature, in the vaginal canal, creates a favorable environment for the lactobacilli flora, positively affecting the microbial population and thus contributing to the prevention of all those problems related to increased vaginal sensitivity to pathogens during "menopause." In addition, recent data show that vitamin E can counteract the production of biofilm produced by certain pathogens, which has detrimental effects on the homeostasis of the vaginal
environment.
The purpose of this non-controlled Investigator Initiated Trail (IIT), with a retrospective design is to evaluate the performance and safety of the vaginal administration of tocopherol acetate in pre-peri and postmenopausal women. The main objectives of the study will be the evaluation of the vaginal pH, of the vaginal eubiosis improving the lactobacilli flora and microbiota, and of sign and symptoms of women. The patients were visited at baseline (with the collection of vaginal swabs) and treated with vaginal administration of tocopherol acetate for 14 days. At the end of treatment (final visit) the patients were visited with the collection of vaginal swabs.
The period of observation is from January 1st, 2020, to December 31st, 2022, while the Data Collection period is planned from March 30th, 2024 to May 31st, 2024.

[STEPS]
TITLE: Copy number variation analysis by ddPCR
AUTHORS: Katie Jing Kay Lam, Olubankole Arogundade, Claire D Clelland

[DESCRIPTION]
This protocol describes copy number variation analysis using Bio-Rad Droplet Digital PCR ddPCR and QuantaSoft Software (with modifications).

[STEPS]
SECTION: ddPCR reaction set up
2. Thaw all components to room temperature
SECTION: Droplet generation
6. Insert DG8‱ Cartridge into cartridge holder

 

SECTION: Droplet generation
7. FIRST - Load >20µL sample into wells
Note:
- Add a 50:50 mix of Supermix & H2O in empty wells
- All 8 wells must be loaded with samples or Supermix + H2O
- Make sure there are no bubbles
SECTION: Droplet generation
8. Load 70 µL of generation oil into all 8 wells
Note:
- Make sure samples are loaded before generation oil
- Generation oil is one-time use/can only be used within a day once opened
SECTION: Droplet generation
9. Apply a DG8 Gasket on cartridge
SECTION: Droplet generation
10. Load cartridge into QX200‱ Droplet Generator

 


SECTION: Transfer Droplets
11. Droplets should be transferred to 96-well plate within 5 mins of generation
SECTION: Transfer Droplets
12. Transfer 40 µL droplets to a ddPCR 96-Well Plate
Note: Pipette SLOWLY for droplet suction and dispensing
SECTION: Transfer Droplets
13. Cover 96w plate with a foil seal sheet
Note: The side with red line should be facing up

 

SECTION: Transfer Droplets
14. Seal plate in a PX1 PCR Plate Sealer at 180°C for 5 secs

 

SECTION: ddPCR cycle
15. ddPCR cycle should be started within 40 mins after sealing
SECTION: ddPCR cycle
16. Place plate into Bio-Rad Thermal Cycler with 96-Deep Well C1000 block

 
 
SECTION: Reading the plate
17. Set plate in room temperature for 1-2mins
Note: The plate can be stored at 4°C for ≤3 days before reading
SECTION: Reading the plate
18. Place plate in Bio-Rad QX200‱ Droplet Reader

 

SECTION: Reading the plate
19. Open QuantaSoft Software & set up


SECTION: Reading the plate
19.1. Select a well
SECTION: Reading the plate
19.2. E.g. Sample
- Enter sample name
- Experiment: CNV2
- Supermix: ddPCR Supermix for Probes (no dUTP)
SECTION: Reading the plate
19.3. E.g. Target 1 (FAM)
- Enter name
- Type: Ch 1 Unknown
SECTION: Reading the plate
19.4. E.g. Target 2 (HEX/VIC)
- Enter name (RPP30)
- Type: Ch 2 Reference
SECTION: Reading the plate
19.5. Repeat (copy & paste) for every well used in the plate
SECTION: Reading the plate
20. Save template & RUN
SECTION: Data analysis
22. Set threshold manually (pink line) - if necessary
 

SECTION: ddPCR workflow
1. 
 

SECTION: ddPCR reaction set up
3. Prepare 20X target primers/probe
 
 
SECTION: ddPCR reaction set up
4. Prepare Master Mix – Number of reactions + 1 (as extra)
 
 
 
SECTION: ddPCR reaction set up
5. For each PCR tube: DNA sample (Up to 350 ng) + Master Mix = 22 µL
SECTION: Data analysis
21. Check if there are >10k events for each sample
TITLE: Mapping ONT Reads to Reference Sequences with Minimap2 and Samtools
AUTHORS: Hung Luong, Hiep Vu

[DESCRIPTION]
This simple protocol maps ONT raw reads to Reference Sequences by Minimap2 and Samtools.

[STEPS]
SECTION: Install requirement software
1. git clone https://github.com/lh3/minimap2
cd minimap2 && make
SECTION: Install requirement software
2. conda create -n samtools
conda activate samtools
conda config --add channels bioconda
conda config --add channels conda-forge
conda install -c bioconda samtools
SECTION: Align reads to reference sequences (two options as bellow)
3. # Without index reference sequence
minimap2 -ax map-ont /path/to/list/references.fasta /path/to/raw/reads.fastq.gz > /path/to/output/folder/alignment_reads.sam
SECTION: Keep only mapped reads
5. samtools view -bS -F 4 /path/to/output/folder/alignment_reads.sam > /path/to/mapped/reads.bam
SECTION: Sort out the mapped reads
6. samtools sort /path/to/mapped/reads.bam -o /path/to/mapped/reads_sort.bam
SECTION: Convert mapped reads to FASTQ or FASTA file for downstream analysis
7. samtools fastq /path/to/mapped/reads_sort.bam > /path/to/mapped/reads_sort.fastq
SECTION: Align reads to reference sequences (two options as bellow)
4. # Index reference sequences first
minimap2 -d /path/to/index/references.mmi /path/to/reference/sequences/references.fasta
# Align reads to reference sequences with ONT reads
minimap2 -ax map-ont /path/to/list/references.fasta /path/to/raw/reads.fastq.gz > /path/to/output/folder/alignment_reads.sam
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A non-invasive PCR-based methodology for sensitive detection and identification of parasitic nematode DNA released in the faeces of infected amphibians and reptiles as egg or tissue fragments (environmental DNA). A DNA extraction protocol optimised for liberation of DNA from resilient parasite eggs was developed alongside the design of a novel, nematode universal, degenerate primer pair that has broad-spectrum detection ability for shed nematode eDNA. </p>

[STEPS]
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TITLE: Rotarod Test
AUTHORS: Sabina Marciano, Roberta Marongiu

[DESCRIPTION]
Behavioral assay to measure motor impairments, specifically balance and coordination, in mice.

[STEPS]
1. Set up rotarod equipment as needed and clean thoroughly.
2. Turn on rotarod using green button and log into computer. Open software directly from desktop once machine is ON and connected.
3. Specify settings:
a.    Protocol: Increasing speed
b.    Speed: 4.0- 40 RPM
c.    File name [EXP NAME].txt                
       i.     Important: save file as .txt to then convert to excel once saved.
d.    Subject ID: animal number 
4. Place mouse on rotarod—can place up to five animals at a time. Make sure mice are all facing the same direction (i.e. wall, AWAY from experimenter) before starting. 
5. Press RESET to start. IMPORTANT. IF YOU DO NOT PRESS RESET, THE SPEED SETTINGS WON’T APPLY.
6. Record behavior for 5 minutes or until mouse falls. If mouse falls <5s, place back on rod, reset zone using button, and continue recording. If mouse stays static (not walking/running but gripping rod) and spins more than three times, remove and mark as failed trial.
a.    IMPORTANT: The sensors register when a mouse falls and stops experiment for that zone. You must remove the fallen mouse as quickly as possible so that it doesn’t intercept another zone. 
7. Place mice back in home cage and leave to rest for one minute before starting next trial. Clean rod in the meantime (within cage, use only water and/or kimwipe. Between cages, clean rod with ethanol and
water).
8. Perform five 5-minute (300 second) trials per cage.
TITLE: U Michigan - Tail Cuff Blood Pressure Determination
AUTHORS: Jeff Hodgin

[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">This protocol provides information for the non-invasive measurement of blood pressure--tail cuff blood pressure determination operated by CODA tail-cuff blood pressure system in mice.</div></div>

[STEPS]
?. Setting up Equipment and Software
1.1  Turn on warming platforms. The ideal temperature for blood pressure (BP) monitoring is 30 ~ 35 ̊C. If the mouse tail is too cold, BP will not be read.However, if the warming platform is too hot, mice will be stressed and may lead to severe dehydration or even death.
1.2  Select appropriate mouse holder and warm the holder, too.
1.3  Turn on the CODA Blood Pressure System and then double-click the icon of CODA program on computer.
?. Mouse preparation
2.1  Gently handle the mouse and place it in the holder (do not force).
2.2  Attach the end piece of the mouse holder carefully in order not to pinch the tail or any other body parts.
2.3  Lead the mouse tail through the O-Cuff first and then through the VPR-Cuff.
2.4  Fasten the cuffs by sliding the hose into the notch on top of the mouse holder.
2.5  Let mouse acclimate and warm in the holders with the tail cuff on for 3-5 minutes and use a blanket to cover mouse tails at the same time.
?. Blood pressure measurement
3.1  In CODA software, select file and choose new, enter mouse information and the names of experiment and researcher under New select Experiment or Session according to the instructions on the screen.
3.2  Click the “Finish” button will begin blood pressure measurements at once.
3.3  Measure blood pressure for 20 cycles each mouse.
3.4  Release the mouse and put them back to the cage immediately.
?. Data collection and processing
4.1  Export the data as an excel sheet to designed folders.
4.2  Measure blood pressure a minimum of 3 days and a maximum of 5 consecutive days at the same time each day
4.3  Calculate the average systolic BP, diastolic BP and mean arterial pressure (MAP) for each mouse.
TITLE: LC-MS/MS quantitation of insulin, glucagon, somatostatin, and C-peptide 
AUTHORS: Matthew J. Donohue, Robert T. Filla, Daniel J. Steyer, Wesley J. Eaton, Michael G. Roper

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">An LC-MS method using multiple reaction monitoring for quantitation of insulin, C-peptide, glucagon, and somatostatin secretion from human islet populations is described. A 5 min separation was developed using a 2.1 × 30 mm (i.d. x length) C18 column. The method was used to measure secretions from static incubations of human islets from 2 donors. Insulin and C-peptide matched well with reported values of these hormones.</div></div>

[STEPS]
TITLE: Fluorescence in situ hybridization (FISH)
AUTHORS: Peter Sims

[DESCRIPTION]
Fluorescence in situ hybridization (FISH) mRNA

[STEPS]
1. Mouse brain sections were prepared, removed from cryoprotectant solution, and washed three times in tris-buffered saline (TBS) at room temperature. 
Sections were incubated with hydrogen peroxide (ACD) for 15 min at room temperature, washed several times in TBS, and then mounted to Superfrost slides. 
Sections were allowed to dry for 10 min and a hydrophobic barrier (PAP pen, Vector Labs) was created around the tissue. 
Tissue was incubated in 50% EtOH, then 70% EtOH, then 100% EtOH for 5 min each. 
Sections were rehydrated in TBS for several minutes, digested withProteaseIV (ACD) for 25 min at room temperature, and rinsed twice with TBS before proceeding to the RNA Scope Multiplex Fluorescent v2 assay (ACD).
2. The RNA Scope Multiplex Fluorescent v2 assay was conducted according to the manufacturer’s instructions, with all incubations taking place in a humidified chamber at 40 °C. 
Two 5-minute washes in excess RNA Scope Wash Buffer (ACD) took place between each incubation in sequential order: probes (2-hours), AMP1 (30 min), AMP2 (30 min), AMP3 (15 min), HRP-C1/2/3 (15 min), TSA Cy3 (30 min), HRP blocker (30 min), HRP-C1/2/3 (15 min), and TSA Cy5 (30 min). 
Samples were washed twice more in RNA Scope Wash Buffer, then twice more in TBS. 
Samples were then blocked and immunostained for tyrosine hydroxylaseas. 
After immunostaining, samples were mounted in Fluoromount G and stored at 4 °C for up to 1 week before imaging.
TITLE: Vandy - Myocardial Ischemia Reperfusion
AUTHORS: Lin Zhong, Jeffrey Rottman, Chee Lim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">The most common cause of cardiovascular mortality in man is the outcome from myocardial ischemic injury. Accordingly, it is necesarry to study the corresponding process of heart injury recovery in many mouse models relevant to human cardiovascular disease. This protocol describes the surgical induction of myocardial injury via  transient occlusion of a coronary artery followed by reperfusion (ischemia-reperfusion injury).</div></div>

[STEPS]
?. Mice are anesthetized with pentobarbital (50 mg.kg, IP).
?. The ventral neck and left parasternal region is shaved and disinfected with Betadine followed by 70% alcohol.
?. The mouse is positioned supineon a heating pad and a small incision is made through the skin underlying the trachea.
?. The trachea is exposed, a small puncture is made in the trachea, and endotracheal intubation is performed using a PE-50 tube.
?. The endotracheal tube is connected to a small rodent ventilator (Harvard Apparatus) for mechanical ventilation of the mouse.
?. ECG electrode leads are placed subcutaneously to monitor the ECG during myocardial infarction(MI).
?. With the use of a surgical microscope, a left thoracotomy is performed and the fourth intercostal space is entered using scissors and blunt dissection.
?. An 8-0 silk suture is placed through the myocardium into the anterolateral left ventricular wall around the left anterior descending (LAD) coronary artery. A sterile PE-50 tube is placed against the LAD and the suture is tied against the tube resulting in ligation of the LAD. During the ischemic period, the surgical area is covered with sterile gauze soaked in warm saline. Following the ischemic period, the tube is removed and the LAD ligature cut resulting in restoration of blood flow and reperfusion.
?. The chest is closed in layers with 7-0 sutures.
?. The mouse is gradually weaned from the ventilator to resolve any possible pneumothorax.
?. Once spontaneous respiration resumes, the endothracheal tube is removed and the trachea incision is closed with 8-0 suture. The skin is then closed with 6-0 suture.
?. The mouse is maintained on the heating pad until fully recovered from anesthesia.
?. Buprenorphineis administered SC immediately following surgery and every 8-12 hr for 72 hr.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<p>This protocol describes the immunostaining of cultured rat hippocampal neurons to stain for ratFwe2 in cell body. It is from 'Flower Ca<sup>2+</sup> channel in CME and ADBE' of Yao CK et al.</p>
<p> </p>
<p>Please see the manuscript for the full method details.</p>
</div>

[GUIDELINES]
<p style="text-align: justify;">To stain for Synaptophysin and mFwe2-HA in presynaptic compartments, the cells were permeabilized with 100% methanol at -20°C for 15 min. 1XPBS solution was used for all staining steps. Primary antibody dilutions used: rabbit anti-GFP, 1:500 (Invitrogen); guinea pig ant-m/ratFwe2 (GP67), 1: 100. rabbit anti-GM130, 1:500 (Adcam); mouse anti-HA, 1:200 (Sigma); mouse anti-SVP38, 1:200 (Sigma). Secondary antibodies were diluted in 1:500 (Jackson ImmunoResearch and Invitrogen). DAPI was used in 1: 2000 (Invitrogen). Images processing was achieved using LSM Zen.</p>
<p style="text-align: justify;"><strong> </strong></p>

[STEPS]
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TITLE: Complex I activity assay
AUTHORS: María José Pérez J., michela.deleidi

[DESCRIPTION]
This protocol describes the complex I activity assay.

[BEFORE_START]
All assays are carried out at 25 °C.
After mitochondrial isolation (Qproteome Mitochondria Isolation Kit. QIAGEN Cat. No. / ID: 37612), resuspend the final pellet in 50 µL of storage buffer, keep isolated mitochondria on ice.
In a ventilated hood, weigh out 6.5 mg of KCN and dissolve in 1 mL of 0.1 Molarity (M) NaOH (stock solution of 100 millimolar (mM))
Label two polystyrene tubes as A and B. For 20 reactions prepare: 
 
      Tube A (1 ml)  Tube B (675 ml)    910 ml of Complex I activity buffer  625 ml of Complex I   activity buffer    20 ml of 100mM KCN (1 mM)  30 ml of NADH assay   reagent    50 ml FF-BSA Assay Reagent  20 ml of Ubiquinone   assay reagent    20 ml of Vehicle       
 








[GUIDELINES]
Reference for analysis: https://www.nature.com/articles/nprot.2012.058

[STEPS]
SECTION: Protocol
1. Distribute the contents of tube A and B in strips suitable for multichannel use. 
SECTION: Protocol
2. In a Half Volume 96-well clear plate add 50 µL of the contents of tube A to each well.

SECTION: Protocol
3. Add 20 µL of sample to each well.

SECTION: Protocol
4. Place plate in plate reader and add 30 µL of B to each well.
SECTION: Protocol
5. Immediately measure absorbance at 340 nm in kinetic read mode (30 seconds intervals for 5 min at 25 °C)

SECTION: Calculations
6. The specific activity of complex I is calculated as nmol min−1 mg−1 of protein according to the following equation:

Enzyme activity (nmol min−1 mg−1) = (Δ Absorbance/min × 1,000)/[(extinction coefficient × volume of sample used in ml) × (sample protein concentration in mg ml−1)].
SECTION: Calculations
7. Extinction coefficient for NADH 6.2 mM-1 cm-1.
TITLE: Assessing Precision of One’s Own Pipetting
AUTHORS: Jonathan Phillips, Gregor Blaha

[DESCRIPTION]
Accurate pipetting is critical for producing precise in vitro assay results. However, since pipetting is such a routine task, many researchers believe their pipetting is precise enough. Rarely do they critically evaluate their own pipetting. This protocol provides an affordable way to assess one's own pipetting. 

[STEPS]
TITLE: Dissolved sulfide concentrations (H2S, HS-, S2-) colorimetric assay using a plate reader (96-well plate)
AUTHORS: Jian Gong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the adaption of a sulfide colorimetric assay originally described in </span><a href="https://doi.org/10.4319/lo.1969.14.3.0454" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Cline 1969</span></a><span>, modified for use on a multi-mode plate reader spectrophotometer (BioTek, Synergy 2, Winooski, VT, USA), using standard </span><a href="https://www.sigmaaldrich.com/catalog/product/SIGMA/CLS9017?lang=en&region=US" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">96-well plates</span></a><span> for rapid measurements of < 400 μL of water samples. This assay measures the concentration of total sulfide species (H</span><span style = "vertical-align:sub;">2</span><span>S, HS</span><span style = "vertical-align:super;">-</span><span>, S</span><span style = "vertical-align:super;">2-</span><span>) in solution.</span></div><div class = "text-block"><span>Samples for this assay should be filtered (</span><a href="https://www.sigmaaldrich.com/catalog/product/aldrich/wha67802502?lang=en&region=US" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">0.2 µm syringe filter</span></a><span>) and placed in </span><a href="https://www.sigmaaldrich.com/catalog/product/sigma/t9661?lang=en&region=US" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">1.5 mL Eppendorf tubes</span></a><span>. Care should be taken to avoid oxidation of the sample by oxygen in the atmosphere, by completing the first step of the assay quickly right after sampling, which stabilizes the sample against further oxidation.</span></div></div>

[STEPS]
?. Sample stabilization: Add 400 µL of liquid sample in 1 ml of reagent A upon sampling. This step makes zinc sulfide and stabilizes the sample from further oxidation (should be already done during sampling). Here 400 µL corresponds roughly to a detection range at 3 - 40 µM sulfide-sulfur. This amount can be further reduced (for example, adjusted to 200 µL) if higher sulfide concentrations are expected. This step dilutes the sample while also stabilizes the sample.
?. Colored-complex development: Transfer 900 µL of the stabilized mixture to a new tube and 15 µL of reagent B is added. Mix by pipetting up and down a few times. The reaction is timed for 20 minutes, in the dark.
?. Measurement: Measure absorbance at 670 nm. The system at the Bosak Lab is a multi-mode plate reader spectrophotometer (BioTek, Synergy 2, Winooski, VT, USA). Measure by loading quadruplicate 200 μl mixtures on a 96-well plate.
TITLE: Keto Start ACV {2022} – Benefits &amp; Side Effects! Is It helpful For Easy Weight Loss?
AUTHORS: Danieldevines  

[DESCRIPTION]
Keto Start ACV can likewise be applied as a wellspring of electricity and a craving suppressant. These chewy sweets are simply handy for purchase at the authority site. Many people take apple juice vinegar to help them with dropping kilos and paintings on their standard wellness. To accomplish your wellbeing objectives, get a field of apple juice vinegar chewy sweets at this moment.

[STEPS]
TITLE: Radioactive Labeling with T4 PNK 3&#39; phosphatase minus (M0236)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Radioactive Labeling with T4 Polynucleotide Kinase 3' phosphatase minus.

[STEPS]
1. Set-up the following reaction:  
 

 

2. Incubate at 37 °C for 30 min.  
3. Heat inactivate by incubating at 65 °C for 20 min.