eamag/protocols-with-steps huggingface dataset:
TITLE: Library positive controls (LPCs) prepared on the Bravo NGS Workstation
AUTHORS: Anna Schmidt, Sarah Nagel, Matthias Meyer

[DESCRIPTION]
Protocol for the preparation of library positive controls (LPCs) in FluidX tubes using the Bravo NGS Workstation. LPCs are used as part of the library preparation workflow of the Ancient DNA Core Unit of the MPI-EVA.

[STEPS]
TITLE: CODEX® Multiplexed Imaging | Tissue Sectioning
AUTHORS: Diane  Saunders, Alexander Hopkirk, Conrad Reihsmann, Regina Jenkins, Marcela Brissova, Alvin C. Powers

[DESCRIPTION]
This protocol describes the tissue preparation processes for the CODEX® (now PhenoCycler™) system by Akoya Biosciences. For the comprehensive multiplexed imaging workflow currently in use at the Vanderbilt Diabetes Research Center, please see CODEX® Multiplexed Imaging | Modality overview.

[STEPS]
SECTION: Coverslip Preparation
1. Gather reagents:

 
 
SECTION: Coverslip Preparation
2. Gently place coverslips at the bottom of a 500-mL glass beaker and swirl to spread the coverslips out. Add approximately 20 mL poly-L-lysine solution to the beaker, ensuring coverslips are fully submerged. Rotate the beaker at a 45° angle for 1 min to mix.
SECTION: Cryosectioning
8. Set the cryostat temperature to -20 °C. Remove a fresh blade, clean with ethanol and a lint free wipe as needed, and insert blade.

SECTION: Cryosectioning
9. Using OCT or CMC, mount sample onto the cryostat chuck in the desired orientation. Trim CMC using larger section increments of 30-50 μm until the tissue becomes visible (Figure 1a).


    
SECTION: Coverslip Storage
13. 3D printed coverslip holders:   

We use these with the following:

  
 
  
SECTION: Coverslip Preparation
4. Slowly pipet off the poly-L-lysine solution and dispose.
SECTION: Coverslip Preparation
7. Spread WypAll® towels on the benchtop. In small batches, remove coverslips from the beaker, separate from one another, and place individual coverslips side by side on towels. Layer another towel on top and gently dab to remove excess liquid. Leave on benchtop until completely dry.

ⓘ Coated coverslips can be stored in a petri dish for up to 2 months.
SECTION: Coverslip Preparation
6.   and repeat for a total of 5-7 washes.
SECTION: Coverslip Preparation
5. Fill the beaker containing the coverslips to half volume with Milli-Q® water. Swirl gently, let sit for 30 s, and then slowly pour off water into the sink.
SECTION: Coverslip Preparation
3. Cover beaker with parafilm and incubate for minimum of 720 min at Room temperature. Coverslips can sit in the poly-L-lysine solution for up to one week.
SECTION: Cryosectioning
10. Obtain   to   sections, using a fine-tipped paintbrush to guide the section onto base and place coverslip onto section (Figure 1b-1d).
SECTION: Cryosectioning
11. Using forceps, slot coverslips with mounted tissue into a plastic coverslip holder under the appropriate donor/block ID label(s).
SECTION: Coverslip Storage
14. Store coverslips at -80 °C for up to 6 months.

SECTION: Cryosectioning
12. As necessary, mount adjacent sections onto Gold Plus slides (for traditional immunohistochemistry) and/or ITO slides (for mass spectrometry). Record all sections cut, the thickness, and any additional block shaved off in the process.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>Modified from the Handbook of Methods in Aquatic Microbial Ecology (p. 220)</p>

[STEPS]
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TITLE: Data review
AUTHORS: Alise J. Ponsero

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols aims to details the steps necessary to review new data points for the "Database Justice League" project.</div></div>

[STEPS]
?. [Data download]
Once you have received the link to download your dataset, download the csv file locally.Make sure to save it on your computer in a convenient folder!The goal of this review process is to Ensure a quality control for the dataset collected by peersDiscuss and document potential choices that have to be taken during the collection process
?. [Database name and link]
First, access the article describing the datasetRemember that you can use the column "DOI" to find the article online.
In order to access the paper search in you web browser :            https://doi.org/ + DOI number
?. [Database name and link]
First, look at the name collected for the database and the one in the paper. Do you see any potential mistakes?Keep in mind the following:Does the database has two names or one? Was this reported correctly in the dataset to review?Is the paper about a new database or an update? Was this reported correctly in the dataset to review?If any disrepency or conflict is detected, add a note in the Note_review2021 column of the spread sheet. Do not modify the original data collected direclty.
?. [Database link and accessibility]
Next we want to assess if the URLand the accessibility reported for the database is exact.Look at the URL collected, is it matching the one visible in the article?If any disrepency or conflict is detected, add a note in the Note_review2021 column of the spread sheet. Do not modify the original data collected direclty.
?. [Database link and accessibility]
Finally, we want to check the availability of the database.Follow the link and assess if the ressource is available or not. Does it match what was reported in the data collected?Once again, if any disrepency or conflict is detected, add a note in the Note_review2021 column of the spread sheet. Do not modify the original data collected direclty.
?. [Submit dataset]
Great job! You collected all the data we needed to complete the DB Justice League! Upload the table on Github and notify us of the upload!Thank you for your great contribution!
TITLE: Advanced Dynamic Weight Bearing system for mice
AUTHORS: Fanglin Lu

[DESCRIPTION]
We can assess spontaneous limb pain-like behaviors using advanced DWB apparatus, wherein changes in the postural equilibrium of each freely moving animal were tracked and analyzed. 
This protocol outlines the procedures from calibration to data analysis. 

 


[BEFORE_START]
Install the DWB software set (drivers for digital USB camera, driver for PC/sensor interface, video codec, advanced DWB software).

[STEPS]
SECTION: Calibration
1. 
 
Connect the sensor and the camera to the PC.

  

SECTION: Data acquisition
7. Place the mouse in the chamber.
Adjust the camera aperture and focus to get a bright and clear image.
Click "Capture" to lauch the acquisition.
Input the weight and click "OK".

 
The mouse is allowed to move freely in the chamber and changes in postural equilibrium were synchronously and automatically tracked and analyzed by the software. 

SECTION: Data acquisition
8. When the capturing is over, click "analysis" and compress the data file.
Place the mouse back to its cage and clean the chamber before the placement of next mouse.
SECTION: Data analysis
9. Click "Menu"→"Open experiment file..."
Manually validate each automatically presumed paw position to avoid error identification afterward.
  
We suggest to choose "never" make a backup during validation as it takes time to save the file.
  
Click "Legend" to help validating each paw position.

  
 

SECTION: Acquisition setting
4.     

SECTION: Calibration
2. Lauch the software and select the sensor type "Mice".
Click "Full calibration".
  
Enter the calibration weight, then click "Next".
For mice, 2000-3000g is preferred, and 7000-10000g for rats
  
Adjust the display angle of the sensor to match the real sensor direction by clicking "<" or ">" on the top.
Make sure no weight is on the sensor and click "Tare".
  
Click "Gain preview", and apply the calibration weight (3000g) indirectly on the sensor.
  

    

SECTION: Calibration
3.   

SECTION: Acquisition setting
5.     
SECTION: Acquisition setting
6. Click "Next".
Click "Video setting" to make sure that the DWB software is connected to the digital camera, not WebCam of the PC. 
Click "OK".
SECTION: Data analysis
10. After validation, click "Result".
   
Click "Graphics" to visualize the results.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Protocol for labeling Gambierdiscus cells with FISH probes in PIG.

[BEFORE_START]
Turn on the incubator and set to correct temperature according to probe.<br />Get probes and Poly A out of freezer to thaw in dark location.<br /><br />

[STEPS]
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TITLE: Perfusion-fixation procedure for adult rhesus monkeys
AUTHORS: Yoland Smith

[DESCRIPTION]
This protocol details Perfusion-fixation procedure for adult rhesus monkeys.

[STEPS]
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
1. Add glutaraldehyde to the fixative solution.
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
2. Start oxygenation of the Ringer’s solution.
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
3. Anesthetize the animal with Ketamine (10 mg/kg, i.m.) or Telazol (3 mg/kg-5 mg/kg, i.m.) in its home cage and bring to the necropsy room.
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
4. Place an i.v. catheter.
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
5. Perform a tracheal intubation.
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
6. Inject 1 mL of heparin (i.v.).

SECTION: Perfusion-fixation procedure for adult rhesus monkeys
7. Inject an overdose of pentobarbital (25 mg/kg i.v.).

SECTION: Perfusion-fixation procedure for adult rhesus monkeys
8. Ensure the absence of all reflexes, including toe pinch reflexes and brainstem reflexes, such as corneal reflexes.
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
9. Cut the thoracic cage to provide access to the heart, clamp the descending aorta, open the pericardium and expose the heart muscle, make an incision of the right atrium and insert the perfusion needle in the left ventricle.
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
10. Artificially ventilate the animal through the tracheal tube during the Ringer’s solution perfusion.
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
11. Infuse transcardially ~300-400 mL of cold oxygenated Ringer’s solution at a rate of 80-90 ml/min through a needle (14G; 1.5 inch long) inserted in the left ventricle. Let the infused solution exit the vascular system through a hole in the right atrium.

SECTION: Perfusion-fixation procedure for adult rhesus monkeys
12. Perfuse 2.5 L of fixative, starting at a rate of 80-90 ml/min for the first liter and then reduce the rate to 50 ml/min for the remaining solution.

SECTION: Perfusion-fixation procedure for adult rhesus monkeys
13. After perfusion, fix the animal’s head in a stereotaxic frame and open the skull using a bone saw and rongeur.
SECTION: Perfusion-fixation procedure for adult rhesus monkeys
14. Cut the brain in  -thick blocks in the coronal stereotaxic plane, remove the resulting blocks of tissue from the skull and post-fix them for 1440 min in 4% paraformaldehyde solution in PB (0.1 Molarity (M), pH 7.4) at 40 °C.

SECTION: Perfusion-fixation procedure for adult rhesus monkeys
15. Transfer the tissue into a phosphate-buffered saline (PBS, 0.01 Molarity (M), pH 7.4) solution.

SECTION: Perfusion-fixation procedure for adult rhesus monkeys
16. Cut the brain in  -thick sections using a vibrating microtome or a freezing microtome and store in a -20 °C freezer in an anti-freeze solution ( Please refer material section) until further processing.
TITLE: DNA Extraction from Bacteriophages - 96 well format
AUTHORS: Frej   Larsen

[DESCRIPTION]
Extraction of viral DNA/RNA from culture lysate or filtered samples free from bacteria. The protocol is based on the Qiagen kit QIAamp Viral RNA Mini kit for 96 well plates and uses consumables from this kit as well as the Pierce Universal Nuclease. 


[BEFORE_START]
Before beginning this protocol, ensure that wash buffers have been properly diluted with ethanol and that the centrifuge is available and not chilled at low temperatures may impede ethanol evaporation.

[STEPS]
1. Pipet 1 µL  100x diluted Pierce Universal Nuclease (2.5units/uL) to each well of a Qiagen S-block

2. Transfer 140 µL of each sample from the outer chamber of the CentrisArt filter tube to the S-block. Mix by pipetting

3. Incubate for 5 min  at room temperature

4. Add 540 µL AVL buffer to inactivate nucleases and lyse phage heads. Mix by pipetting

5. Incubate 10 min  at room temperature

6. Add 560 µL  absolute ethanol. Mix thoroughly by pipetting

8. Transfer 630 µL  to the QIAamp 96 plate, then seal it with an AirPore tape sheet

7. Place a QIAamp 96 plate on a new S-block
9. Load the QIAamp 96 plate on the S-block into a rotor bucket. Centrifuge  5500 x g, 4 min, 21 °C or until all liquid has passed through the filter

10. Repeat steps 8 and 9 until all of the sample has passed through the filter

11. Replace the bottom S-block and add 500 µL  AW1 wash buffer to each well, then seal the QIAamp 96 plate

12. Load the QIAamp 96 plate on the S-block into a rotor bucket. Centrifuge  5500 x g, 5 min, 21 °C or until all liquid has passed through the filter
13. Remove the seal, add 500 µL  AW2 wash buffer to each well, then reseal the plate.

14. Load the QIAamp 96 plate on the S-block into a rotor bucket. Centrifuge  5500 x g, 5 min, 21 °C or until all liquid has passed through the filter
15. Place the QIAamp 96 plate on a new S-block and centrifuge  5500 x g, 10 min, 21 °C  to dry the membrane
16. Discard the S-block and place the QIAamp 96 plate on an A&A receiving plate. Add 60 µL  AVE elution buffer directly onto the filter membrane 

17. Seal the plate with an AirPore Tape sheet, then incubate at room temperature for 10 min 

18. Load the QIAamp 96 plate on the receiving plate into a rotor bucket. Centrifuge  5500 x g, 4 min, 21 °C 
19. Discard the QIAamp 96 plate, seal the receiving plate for storage and store at -80 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Isolated astrocyte cell culture preparation. Dissociation, preparation and plating of mice cortex neurons and glia cells on MEA. Isolatioin process of the culture from neurons, to obtain an isolated astrocyte culture.

[STEPS]
SECTION: Sample collection
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SECTION: Cell lysis
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SECTION: Cell preparation
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SECTION: Cell culture
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TITLE: Using the Confocal Microscope with Enterococcus faecalis and NAO dye.
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Using the Confocal Microscope with </span><span style = "font-style:italic;">Enterococcus faecalis </span><span>and NAO dye.</span></div></div>

[STEPS]
?. [Steps]
Prepare an Enterococcus faecalis overnight culture.
?. [Steps]
Next morning, dilute overnight E. faecalis to an OD600 of 0.01 in fresh BHI.
?. [Steps]
Check OD600 after 3 hours.
?. [Steps]
Isolate 4mL of log phase culture and place in BD Facs tube.
?. [Steps]
Centrifuge 3500 RPM 5 to10 minutes.
?. [Steps]
Suspend pellet in 1mL of sterilized 1X PBS.
?. [Steps]
Stain with NAO dye for 30 minutes in the dark at room temp.Use 7.5uL of a 10-3diluted NAO stock to give stain with 1.5uM NAO.The initial concentration of NAO is 100mg in 1mL of DMSO or 212mM in 1mL of DMSO.Make a 1000 fold dilution in DMSO 1uL → 999uL DMSOThis dilution is stable if kept wrapped in tin foil at room temp.
?. [Steps]
After 30 minutes, flood stained cells with 3mL of 1X PBS.
?. [Steps]
Centrifuge 3500 RPM 5 to10 minutes.
?. [Steps]
Suspend pellet in 4 mL of 1X PBS.
?. [Steps]
Centrifuge 3500 RPM 5 to10 minutes.
?. [Steps]
Suspend pellet in 15uL of 1X PBS.
?. [Steps]
Prepare slides with a poly-L-lysine fixative
?. [Steps]
Add 9uL of stained cells to the fixative.
?. [Steps]
Add a drop of Anti-Fade reagent
?. [Steps]
Place a coverslip.
?. [Steps]
Seal coverslip with VALAP.  Smear it around the edges.
?. [Steps]
Observe on Confocal.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Atrial fibrillation (AF) is the most common arrhythmia. The most common diagnostic method, 12-lead ECG, can record episodes of arrhythmia from which the type and severity can be determined. The Heart Spectrum Blood Pressure Monitor (P2, OSTAR Meditech Corp.) is used to measure cardiovascular pressure change, with fast Fourier transform (FFT) analysis, to obtain the heart rate frequency variability and accurate blood pressure data. Here we compared the diagnostic efficacy of the Heart Spectrum Blood Pressure Monitor with a 12-lead ECG in patients with AF. Three measurement methods were used in this study: blood pressure, mean arterial pressure (MAP) which is calculated from individual blood pressure as constant pressure, and a constant pressure 60 mmHg, in order to analyze the heart index and compare it with simultaneous 12-lead ECG. The physician used a 12-lead ECG and the Heart Spectrum Blood Pressure Monitor simultaneously. The Heart Spectrum Blood Pressure Monitor used an FFT analysis for the diagnosis of AF; the findings were compared with the readings from the 12-lead ECG. This unblinded clinical trial was conducted at the Emergency Room of Taipei Medical University Hospital. Twenty-nine subjects with AF and 33 without AF, aged 25 to 97 years (mean: 63.5 years), were included. Subjects who had been exposed to high frequency surgical equipment during testing, those with cardiac pacemakers or implantable defibrillators, and pregnant women were excluded. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) are 96.67%, 96.88%, 96.67% and 96.88% for Method 1, 90%, 100%, 100% and 91.43% for Method 2, and 100%, 93.75%, 93.75% and 100% for Method 3, respectively. The sensitivity, specificity, PPV, and NPV for all these methods ranges between 90% and 100%, indicating that the Heart Spectrum Blood Pressure Monitor can be effectively applied to AF detection.</p>

[STEPS]
TITLE: Protocols for Bioinformatic Tools
AUTHORS:  

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this lab, students will work through BLAST and the UCSC Genome browser to find and analyze information about their genes of interest. </div></div>

[STEPS]
?. [BLAST]
BLASTDo a BLAST search of a gene of interest.
?. [UCSC Genome Browser]
What diseases are associated with this gene?
?. [UCSC Genome Browser]
What Biochemical and Signaling Pathways is this gene involved in? Again, what is the signficance?
?. [UCSC Genome Browser]
What orthologs does this gene have? What is the significance of having these as an ortholog?
?. [UCSC Genome Browser]
Look through the tracks given on the displays and give the name of track which is used to give gene expression for different tissue types. What tissue do we observe the highest expression of RNA? (use track other than NCBI RefSeq)
?. [UCSC Genome Browser]
Genome BrowserYou will now have the opportunity to utilize these tools yourself to investigate a gene that you find interesting. Use these tools to answer the questions below and then relay these in the Lab Results of this Lab Notebook.Find a gene of interest and type it onto the genome browser. Use the human GRCh37/hg19 Assembly. In your own words, give a brief description of this gene’s function.
?. [BLAST]
Look at the BLAST results. Give the:Top 5 scoresTop 5 species this gene is found inLowest E-Value (if any)
TITLE: Stereologic Cell Population Estimates in Cleared Human Brain Tissue
AUTHORS: Bridget Wicinski, Patrick.hof

[DESCRIPTION]
We provide a step-by-step protocol for stereologic assessment in the human cerebral cortex using cleared postmortem specimens with a laminar level of analysis for distinct neuronal populations identified by immunolabeling for specific cytoplasmic markers. Stereologic tools provide a set of simple rules and formulas to estimate numerical quantities of various morphologic parameters in tissue, with precision, accuracy and a design free of observer-induced bias. Parameters such a number, density, volume, surface area, or length can be easily estimated based on the systematic sampling of a region of interest (e.g., a layer of a cytoarchitecturally defined region of cortex, defined as a “volume of reference”), with an observer-independent random design, that gives each object under study (e.g., a neuronal population identified by a specific protein marker) the same probability to be sampled once and only once in its volume of reference, based on strict sampling criteria that are kept constant for a given object throughout the analysis. Stereologic analysis was performed on cleared slice from large specimens encompassing Broca’s area (Brodmann’s area 44/45), up to 4x4x2 cm, imaged at 3x3x3 µm pixel dimensions, and immunostained for NeuN, calretinin, and somatostatin, using the MBF Bioscience Stereo Investigator Cleared Tissue software (version 2022.1.1) with an Optical Fractionator design. Data were recorded as estimates of total number of neurons as well as neuronal densities in each layer. The protocol provides the detail of the sampling parameters including counting frame size, grid size, and disector height, definition and contouring of cortical layers and use of their boundaries to estimate laminar surface areas and volume. Full details on software operation and sampling design are explained. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is the protocol for the preparation of denaturing polyacrylamide gels and silver staining</p>

[BEFORE_START]
<p>Prepare:</p>
<p> </p>
<p>FORMAMIDE for Acrylamyde (10mL)</p>
<p> </p>
<ul>
<li>Formamide 9,8mL<br />EDTA 0,5M pH 8,0 125µL<br />Azul de Bromofenol 1µL<br />Xilenocianol 1µL<br />1. Vortex in a falcon.<br />2. Store in a refrigerator.</li>
</ul>
<p> </p>
<ul>
<li>POLYACRYLAMIDE 6% (1L)<br />Acrylamide 40% 150mL<br />TBE 5X 200mL<br />Urea 420g<br />MilliQ Water qsp 1 L<br />1. Place 200mL of milliQ water together with the TBE and Urea.<br />2. Dissolve in shaker under gentle heating.<br />3. Add 40% Acrylamide.<br />4. Complete for 1L.<br />5. Filter with paper filter and store in refrigerator.</li>
</ul>
<p> </p>
<ul>
<li>BUFFER FORMAMIDE + BLUE DEXTRAN (600μL)<br />Blue dextran 100μL<br />Formamide 500μL</li>
</ul>
<p> </p>
<ul>
<li>Blue Dextran (1mL)<br />Blue Dextran 50mg<br />0.5M EDTA (pH 8.0) 500μL<br />Adjust the volume to 1mL and shake until dissolving (stock: 4ºC).</li>
</ul>
<p> </p>
<ul>
<li>BIND<br />Ethanol-Acetic Acid Solution 1mL<br />Bind 3.5μL<br />1. Place the Ethanol-Acetic Acid Solution and the Bind in an eppendorf of 1.5.<br />2. Mix lightly.<br />3. Spread on the plate with a sheet of toilet paper.<br />Note: Prepare this solution on time, just leave the Solution Ethanol-Acetic Acid solution ready.</li>
</ul>
<p> </p>

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