eamag/protocols-with-steps huggingface dataset:
TITLE: UC Davis - Aspartate Aminotransferase
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
Aspartate transaminase (AST) also known as aspartate aminotransferase or (sGOT) is a metabolic enzyme expressed primarily in the liver. Elevation of AST levels is an indication of liver damage and has been associated with liver injury. AST levels are monitored routinely in patients with liver diseases. AST is also a very useful tool for preclinical investigation of experimental drug formulations and AST levels are commonly used to monitor and attenuate the hepatotoxic effects of experimental drugs in rodents.</div></div>

[STEPS]
?. Add 5 μl of each sample or standard (in duplicate) to the microplate wells.IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader.
?. Add 50 μl of AST Reagent Solution to the wells. Cover wells with the adhesive film and incubate at 37°C for 10 min.
?. Carefully remove adhesive film and add 50 μl AST Color Reagent to the wells. Use second film to re-cover wells and incubate for 10 min at 37°C.
?. Remove adhesive and add 200 μl 0.1 M HCl to each well. Read 510 nm absorbance in plate reader.
?. A standard curve can be constructed using the serially-diluted standards by plotting the average absorbance for each oxaloacetate standard against its concentration in U/l. Calculate unknowns from standard curve.
TITLE: Brain Clarification for Neuromelanin visualisation by OPT
AUTHORS: Ariadna Laguna, Miquel Vila

[DESCRIPTION]
Protocol for the clarification of whole mouse brains to allow neuromelanin visualisation by Optical Projection Tomography

[STEPS]
SECTION: Brain processing
1. Mice are transcardially perfused in 4% PFA and postfixed in the same fixative overnight
SECTION: Brain processing
2. After rinsing several times in PBS, brains are embedded in 1% low melting point agarose in water 
SECTION: Brain processing
4. Once dehydrated the brains are incubated during 4 hours shaking in 66% dichloromethane (DCM) /33% methanol at room temperature
SECTION: Brain imaging
8. The brains are scanned with transmission light with three different filters (a cyan fluorescent protein -CFP-filter: emission 460-500nm, a green fluorescent protein -GFP- filter: emission 500-550nm and a DSR filter: emission >610nm) using an OPT imaging system mounted on a Leica MZ 16 FA microscope
SECTION: Brain processing
5. The brains are incubated with shaking in 100% DCM 30 minutes twice to complete the delipidation process
SECTION: Brain processing
7. The brains are subsequently immersed in a BABB-filled chamber for Optical projection tomography (OPT) imaging
SECTION: Brain processing
6. The brains are chemically cleared with BABB (a combination of 1 part of benzyl alcohol and 2 parts of benzyl benzoate)
SECTION: Brain imaging
9. The brain images are visualised using the open source software Fiji
SECTION: Brain processing
3. Brains are dehydrated in 100% methanol covered in aluminum foil to protect the specimen from light; change the methanol three times during the next 24 h or until the specimen is completely dehydrated
TITLE: Measuring the Action of Oligonucleotide Therapeutics in the Lung at the Cell Type-Specific Level by Tissue Disruption and Cell Sorting (TDCS)
AUTHORS: Helen Graves, Steven Evans, Michael Fauler, Manfred Frick, Sterghios A. Moschos

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The clinical potential of DNA and RNA-targeting therapeutics for airways disease has been hampered by the poor translation of promising drug candidates from cell culture to in vivo models and the clinic. For example, classical preclinical approaches routinely report 20–60% target knockdown effects in the lung, where 1 or 2 log effects are observed in isolated cell cultures in vitro</span><span style = "font-style:italic;">. </span><span>Preparation of monocellular suspensions of tissues by mechanoenzymatic disruption followed by cell sorting (TDCS) after in vivo drug dosing, however, can offer pharmacokinetic and pharmacodynamic insights on the effects of drugs to precise cell subpopulations. Moreover, this can be reliably achieved with up to 66% fewer animals than standard in vivo pharmacology approaches due to lower data variance afforded through analytics on defined, viable cell numbers. Here we describe the TDCS methodology for the isolation of total lung epithelia, lung macrophages, and epithelium/macrophage-depleted cell fractions from mouse lungs using a two-stage sorting process of immunomagnetic bead separation followed by flow cytometric sorting using fluorescent antibodies against well-established surface markers such as F4/80, CD11b, and CD326. Validated antibodies for additional cell types and markers are also provided.</span></div></div>

[STEPS]
TITLE: Vascular perfusion of mice
AUTHORS: Peter Mundel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol describes the procedure for perfusion of mice prior to organ or tissue harvesting .</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complications:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakksn.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakksw.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakks6.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakkte.jpg" /></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakktn.jpg" /></div></div>

[STEPS]
?. Preparation (see figure below for setup)♦ Fix tubing (small diameter tubes, below 1 cm) to the bottles: 1 bottle for PFA, 1 bottle for sucrose
♦ Fill perfusion solutions in the bottles: solutions must be freshly prepared and filtered (using 0.22 μm filter)♦ Adjust perfusion pressure to 150-160 mm Hg
♦ Fix needle at the end of tubing after valve: butterfly 23 gauge 3/4
♦ Remove any air from all tubing!!♦ Anesthetic: ketamine-xylazine mix (150/10 mg/kg=0.1 ml/20 g BW)
♦ Mix 3 ml ketamine 100mg/ml + 1 ml xylazine 20mg/ml + 6 ml sterile saline
♦ Fix the anesthetized animal in a plastic tray♦ Solution #1: 4 % PFA in PBS, ph 7.4
♦ Solution #2: 18% Sucrose in PBS, ph 7.4
?. Perfusion:♦ Swab mouse with 70% alcohol to wet the fur, cut open abdominal skin by a longitudinal incision
♦ Remove skin and gut to expose abdomal aorta and Vena cava
♦ Clean the aort abdominalis and vena cava from connective tissue and fat with cotton swabs♦ Clamp aorta abdominalis and vena cava in the area of the iliac arteries
♦ Insert the butterfly 23 gauge 3/4 needle in the Aorta below the renal arteries
♦ Immediately cut open the Vena cava with small and sharp scissors: this should allow a rapid drain of the perfusion solutions!♦ Watch the needle carefully during the entire perfusion!!!
♦ Start perfusion for 3 minutes with PFA, pH 7.4 at 37ºC
♦ Switch valve to Solution #2:♦ Without any interruption of pressure/flow perfuse for 5 minutes with sucrose, 7.4 at 37ºC
♦ Stop perfusion, remove butterfly
♦ Cut out kidneys at renal hilum for further processing
♦ Before starting to perfuse the next animal rinse the tubing and needle extensively
?. Schematic of custom-made perfusion system
TITLE: Oxidative Stress Cell Model 
AUTHORS: Veerle Baekelandt

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = ":UNDERLINE;">Oxidative Stress Cell Model preparation for intact cell cross-linking</span></div></div>

[STEPS]
?. seed cells for  in a 10 cm dish at a density of 6 x 106 cells per plate
?. treat cells for   with 5 mM freshly prepared FeCl2 and 100 µM H2O2 in DMEM-complete (filtered trough a .45µM filter)
?. wash cells with 1X PBS and scrape cells for immunoblotting or intact cell cross linking
TITLE: Yeast competent cells
AUTHORS: Marijn Ceelen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol to make yeast competent cells, which can be then stored at 4 ºC for up to a week. </div></div>

[STEPS]
?. Pick a yeast colony from a plate and place it inside 10 mL YPD medium.
?. Incubate the cells overnight at 30 ºC and 180 rpm.
?. The next day, measure the OD600 of the liquid culture.
?. Inoculate a flask with 50 mL YPD medium with enough volume of the overnight culture to reach an OD600 of 0.4.
?. Incubate the cells for 3-4 hours at 30 ºC and 180 rpm until an OD600 of 2.0 is reached.
?. Place the 50 mL of liquid culture inide a 50 mL tube and centrifuge the cells at 3000x g for 5 minutes and 20 ºC.
?. Discard the supernatant and resuspend the cells in 25 mL of sterile MQ water. Centrifuge cells at 3000x g for 5 minutes and at 20 ºC. Repeat this step another time.
?. Discard the supernatant and resuspend the cells in 1 mL sterile MQ water. Transfer the volume into a 1.5 mL tube.
?. Centrifuge the cells at 3000x g for 5 minutes and discard the supernatant.
?. Resupend cells in 1 mL sterile MQ water and aliquote them by 100 µL in different 1.5 mL tubes. Each tube can be used for a transformation and may be stored at 4 ºC for up to a week.
TITLE: NAO staining
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">NAO staining protocol - Harp - 2014</div></div>

[STEPS]
?. [Steps]
Incubate Enterococcus faecalis at 37oC overnight in 10mL of Brain Heart Infusion (BHI) broth.
?. [Steps]
The next morning check OD600 of overnight culture. Should be around ~1.5.
?. [Steps]
Diluted overnight culture to 0.01 using fresh 10mL BHI broth.
?. [Steps]
Add supplements as necessary.
?. [Steps]
Incubate at 37oC without shaking.
?. [Steps]
After ~3 hours check OD of cultures.Looking for mid-long phase OD600 ~0.4.Bile will need its own dilution blank.Supplements may need more than 3 hours of incubation.
?. [Steps]
Once the cultures have hit OD600 ~0.4, centrifuge cells at 4oC for 5 minutes at 3500 RPM.
?. [Steps]
Re-suspend in equal volumes of 1x PBS.
?. [Steps]
Take a 1mL aliquot of re-suspended culture and stain with 12.5ug of 10-2 NAO (100mg in mL) = 12.5uL.
?. [Steps]
Incubate at RT for ~30 minutes in the dark.
?. [Steps]
Flood 1mL of cells/NAO with ~4mL of 1xPBS. Centrifuge at 4oC for 5 minutes at 3500 RPM.
?. [Steps]
Pour off supernatant - flood with 1x PBS - centrifuge.
?. [Steps]
Re-suspend pellet with 20uL of 1x PBS.
?. [Steps]
Take 8uL of stained cells and put on slide - take to microscope.
TITLE: Vandy – IP glucose tolerance test
AUTHORS: Louise Lantier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">The glucose tolerance test measures the clearance of an intraperitoneally injected glucose load from the body. It is used to detect disturbances in glucose metabolism that can be linked to diabetes or metabolic syndrome. Animals are fasted for 5 hours, fasted blood glucose levels are determined before a solution of glucose is administered by intra-peritoneal (IP) injection. Subsequently, the blood glucose level is measured at different time points during the following 90 minutes. </div></div>

[STEPS]
?. Weigh the mice. For mice of differing fat mass, we recommend dosing the bolus of glucose on lean mass. This requires body composition.
?. Fast mice for 5 hours by transferring mice to individual cages or containers.
?. Prepare an experiment record sheet, sticks for glucose measurement and syringe for IP injection of the Dextrose solution.
?. Calculate and record the volume of 20% Dextrose solution required for each individual mouse for IP injection as follows: a. To inject 2g of dextrose/kg body mass, the volume of the IP glucose injection is: 20% Dextrose (μl) = 10 x body weight (g)
?. Cut the tip of the tail using clean surgical scissors. A small drop of blood (
?. Collect 40µL of whole blood in a tube for measurement of insulin. This is greatly facilitated by use of a collection tube such as Sarstedt’s Microvette® CB 300 (cat # 16.443.100 for Heparin or cat # 16.444.100 for EDTA). Spin the blood and collect plasma in a clean tube (20µL). Store plasma on ice. Spin the blood 1min at 13k rpm and collect plasma in a new tube (25µL).
?. Inject the mouse intraperitoneally with the appropriate amount of glucose solution, as previously determined (point 4) and start the timer.
?. The blood glucose levels are measured at 5, 10, 15, 20, 30, 45, 60, 90 and 120 minutes after glucose gavage. Samples for insulin determination (20µl plasma) are collected at 10, 30, 60 and 120min (Table 1). For each sample, start the bleeding again by removing the clot from the first incision, massage the tail if blood flow is inadequate. Place a small drop of blood on a new test strip. Results are recorded in the record sheet.
?. Ensure that further blood loss from the incision is minimal by briefly applying pressure to the incision after each measurement.
?. At the end of the experiment, mice are euthanized for tissue collection or placed in a clean cage with water and food available ad libitum for recovery. Monitor the mice carefully to observe any abnormal behavior. Plasma samples are stored in -20 or -80ºC freezer until analysis.
TITLE: Bioinformatic workflow for NGS data control
AUTHORS: Khalid  El Moussaoui

[DESCRIPTION]
Workflow for data integrity and quality control of high throughput sequencing on Illumina NovaSeq6000. The analyses are performed on macOS Monterey 12.3.1 running on an ARM-architected Apple Silicon processor. This workflow considers that the user directory (~/) is structured as seen in the "work environment configuration" protocol. To avoid error messages, please follow this protocol and set up your computer before starting.

[STEPS]
SECTION: Activation of the environment
1. Open a terminal window.
 

SECTION: Activation of the environment
2. Activate the previously created QC_env environment by typing the following command in the terminal :
 

SECTION: Data integrity check
3. Considering that the .gz archive downloaded from the GIGA servers has been unzipped under ~/fastq_files, that the original_md5.txt file has been stored under ~/md5 and that the python & R scripts previously created are stored under ~/KE_utilities, type the following command in the terminal to recompute the md5 hash and store it in a new file under ~/md5
 

SECTION: Data integrity check
4. After generating the ~/md5/recomputed_md5.txt file, type the following command in the terminal to launch the python script that allows the data integrity check :
 
This script can be downloaded on GitHub : https://github.com/elmoussaoui-k/drylab_workflow/blob/b41c0a03d7a3fdb023a94b331cab5460d706b6c8/data_integrity_checker.py
SECTION: Data integrity check
5. Specify the path to the original_md5.txt file and then to the recomputed_md5.txt file :
 

SECTION: Run fastQC
6. Start the fastQC analysis on all existing files in the ~/fastq_files directory in recursive mode using "*". Moreover, the addition of the --outdir option allows to specify an output directory for the reports generated by fastQC. This generates an individual .html report for each file.
 

SECTION: Run multiQC
8. To summarize the reports generated with fastQC into a single report, run multiQC. To do this, type the following command in the terminal :
 

SECTION: Filter reads with fastp
10. The reads can be filtered automatically with fastp. Just launch the program, specify the 2 .fastq.gz files (R1 and R2) as input and specify the name and location of the 2 processed files. Adding the -h option allows to specify a folder for the HTML report. The option -j " " allows to cancel the creation of the JSON report. The -R option allows to give a name to the generated HTML report.
 

SECTION: Run fastQC
7. The generated reports can be opened by typing the following command in the terminal :
 

SECTION: Run multiQC
9. The generated report can be opened by typing the following command in the terminal :
TITLE: Reynolds&#39; Lead Citrate Stain
AUTHORS: Jens Berndtsson

[DESCRIPTION]
Reynolds Lead Citrate Stain for electron microscopy is a standard stain developed by Edward S. Reynolds and consists of a mixture of 80 mM Lead(II) nitrate and 120 mM Sodium citrate. Sodium hydroxide is added to make the stain basic to pH 12.

Preparation time:
40-60 min

Reference:
Edward S. Reynolds; THE USE OF LEAD CITRATE AT HIGH pH AS AN ELECTRON-OPAQUE STAIN IN ELECTRON MICROSCOPY .J Cell Biol1 April 1963; 17 (1): 208–212. doi:https://doi.org/10.1083/jcb.17.1.208

[BEFORE_START]
Book a fume hood and label the syringes for aliquots.

[GUIDELINES]
This protocols aim to show the exact method that the Centre for Cellular Imaging at the University of Gothenburg use to prepare a standard buffer.

We do not claim to have discovered, invented or otherwise hold any form of intellectual property of the buffer(s) described in this protocol.

Feel free to visit us: https://www.gu.se/en/core-facilities/centre-for-cellular-imaging

[STEPS]
SECTION: 50 ml Lead citrate for Array tomography
1. Weigh out and dissolve Lead (II) nitrate and tri-Sodium citrate dihydrate:
Weigh out 1.32 g of   in a 50 ml volumetric flask in which you have added a small magnetic stirrer.
Dissolve the Lead(II) nitrate by adding 30 mL of   and turning on the stirrer.
Weigh out 1.76 g of   in a small weighing boat. Add it to the dissolved Lead(II) nitrate.
Shake the mixture vigorously, or vortex it for 1 min The solution will be milky.
Cover the volumetric flask with parafilm and let it stir for an additional 30 min 

Prepare a 1 M sodium hydroxide (NaOH) solution:
Add 10 mL of   to a 15 ml falcon tube. 
Weigh out 0.4 g of   on a small weighing paper, and add the NaOH to the water.
Mix the NaOH until it dissolves by inverting the falcon tube.
 

Finalizing the Lead citrate solution:
Add 8 mL of the 1 M NaOH solution to the Lead(II) nitrate/Tri-Sodium citrate mixture, this will raise the final pH to 12±0.1.
Keep stirring until the solution is clear.
Add   until the volume is 50 ml.

Aliquote the mixture in:
5 x 10 mL - 10 ml syringes labelled: Lead citrate and date.

Storage:
Mix on the day of use for optimal staining. For longer storage, seal syringes with parafilm and store long term (4 weeks) at 4 °C
 

Clean up:
Rinse the 50 ml volumetric flask and magnetic stirrer twice with water, pouring the wash of in a liquid lead waste. Then rinse thouroghly with water before before putting them in the dishwasher.
TITLE: Leaf Protein Extraction for Immunoblot (Soybean, Cowpea, Tobacco)
AUTHORS: Steven J Burgess

[DESCRIPTION]
This protein extraction protocol was developed for analysis of protein abundance in leaf tissue by immunoblot. It was optimized for dicot species including Glycine max.

Quantities - 450 µL protein extraction buffer (PEB) per three 13.4 mm diameter leaf disks (size #7 Humboldt Cork Borer)

Note from Agrisera: Keeping sample volumes in a range of 0.2-0.5 mL has been found to contribute to better extraction results, an upscale in volume is not recommended, if no cork borer available the suggestion is 500 µL PEB per 100 mg of plant tissue. 
- Final volume ~400 µL 
- Expected yield: 1.5 - 6 µg / µL 
- Total yield: 6 - 24 mg protein 
- Amount of total soluble protein to load per lane 30 µg  

Note: when using the TissueLyser II it is recommended to use 2 mL centrifuge tubes in conjunction with 4mm beads. 1.5mL tubes are narrow at the end and samples will not grind properly if you use the 4mm beads.

[BEFORE_START]
Collect tissue immediately into liquid nitrogen and store at -80C.  Grind per protocol "Grinding Tissue with the Qiagen Tissuelyzer".

Prepare a working solution of 1x Protein Extraction Buffer

1x PEB (10 mL) (2.5 mL 4x PEB; 250 µL 2-mercaptoethanol; 100 µL protease inhibitor cocktail; 7.3 mL dH2O)


[STEPS]
SECTION: Buffer Preparation
1. Incubate 4x PEB (if stored at 4 oC) at 50 °C for 20 min to re-suspended precipitated SDS in buffer. 
SECTION: Buffer Preparation
2. Make 500 µL 1x PEB per sample by diluting 4x stock with dH2O. 
 


SECTION: Dissolve ground powder
3. Add 450 µL 1x PEB to ground powder (3x 1cm leaf disk). 

SECTION: Dissolve ground powder
4. Vortex immediately (maximum speed) to mix. Approximately 1 min 
 


SECTION: Dissolve ground powder
5. Heat samples at 95 °C for 5 min  to denature proteins and inactivate proteases.

 

SECTION: Dissolve ground powder
6. Vortex heated samples to shear nucleic acids (5 s ; max speed)

SECTION: Dissolve ground powder
7. Spin samples for 3 min  at  10000 x g to pellet insoluble material
 


SECTION: Dissolve ground powder
8. Transfer supernatant to a new 1.5 mL centrifuge tube and transfer to ice or store at -20 °C for up to a month
TITLE: NCEM Drop - Cell Pellet Dounce Homogenisation (TM-014)
AUTHORS: sandra.crameri

[DESCRIPTION]
cell pellet dounce homogenisation

[STEPS]
SECTION: HEADER
1. SAN:




SPEC No:




OPERATOR:



SECTION: Dounce Homogenisation
2. Remove cells from the flasks by scraping with a disposable plastic scraper and poor into a 10 mL centrifuge tube. 

SECTION: Dounce Homogenisation
3. Pellet the cells in a bench top centrifuge at 2000 rpm 2 min , remove supernatant.

SECTION: Dounce Homogenisation
4. Resuspend the cells in equal volume TC water and transfer to glass homogeniser tube.
SECTION: Dounce Homogenisation
5. Insert teflon plunger into cordless drill, set drill on max speed.
SECTION: Dounce Homogenisation
6. Homogenise(20 strokes) inside Class II BSC cabinet.
SECTION: Dounce Homogenisation
7. Clarify resultant solution at 13000 rpm  in Eppendorf centrifuge for 1 min 


SECTION: Dounce Homogenisation
8. Stand for 5 min to permit viruses to diffuse back into solution, from the debris, and provide an interface for the sampling of membrane associated viruses. Use supernatant for sample below.

SECTION: Conventional
9. Adsorb 10 µL sample to grid 10 min , inspect to ensure sample does not dry out.

SECTION: Conventional
10. Drain excess sample from grid using filter paper, leave wet.
SECTION: Conventional
11. Stain  1 min 

12. Drain & dry using filter paper
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes how to check the quality of modified QCM-D sensors and how to study protein interactions using QCM-D sensing.

[BEFORE_START]
All QCM-D measurements should be performed with an E4 Q-Sense instrument. All protein interaction experiments should be performed in duplicates, under a continuous flow of 50-100 µl/min and with a negative control for which PBS is flown instead of the other reagents.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: PONE-D-23-30545 Stata codes
AUTHORS: Fengming Chen, Hayato Nakanishi, Yoichi Sekizawa, Sae Ochi, Mirai So

[DESCRIPTION]
Fig1. RD plot of the probability of receiving the vaccine twice.
Table 2. Estimation Results of the Regression Discontinuity Design on Outcomes.
S3 Table. Estimation Result of the Effect of Eligibility on Vaccination Rates (Twice).
S4 Table. Estimation Results of the Effect of Eligibility on Vaccination Rates (Treatment: At Least Once).
S5 Table. Estimation Results of Regression Discontinuity Design on Outcomes(Treatment: At Least Once). 
S6 Table. Estimation Results of Regression Discontinuity Design on Outcomes (the First Survey Round).
S1 Fig. Distribution of birth months.

[STEPS]