eamag/protocols-with-steps huggingface dataset:
TITLE: Electrochemical Gradients Assessment Device (EGAD)
AUTHORS: Jack Cerchiara, Kerry Kim, Eli Meir, Mary Pat Wenderoth, Jennifer H. Doherty

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The basis for understanding neurophysiology is understanding ion movement across cell membranes. Students in introductory courses recognize ion concentration gradients as a driving force for ion movement but struggle to simultaneously account for electrical charge gradients. We developed and validated a 17-multiple-choice item assessment of students’ understanding of electrochemical gradients and resistance in neurophysiology, the Electrochemical Gradients Assessment Device (EGAD). We investigated the internal validity of the assessment by analyzing item characteristic curves of score probability and student ability for each question, as well as a Wright Map of student scores and ability. We used linear mixed-effect regression to test student performance and ability as well as learning gains. Our assessment discriminated students with average ability (</span><span style = "font-style:italic;">WLE</span><span> -2 to 1.5 Θ), however, was not as effective at discriminating students at the highest ability (</span><span style = "font-style:italic;">WLE</span><span> > 2 Θ). We determined the assessment could capture changes in both assessment scores (model r</span><span style = "vertical-align:super;">2</span><span> = 0.51, p<0.001, n=444) and ability estimates (model r</span><span style = "vertical-align:super;">2</span><span>= 0.47, p<0.001, n=444) after a simulation-based lab and course instruction for 222 students. Our Differential Item Function analysis determined that each item on the assessment performed equitably for all students regardless of gender, race/ethnicity or economic status. Overall, we found that males scored higher (r</span><span style = "vertical-align:super;">2</span><span> = 0.51, p=0.014, n=444) and had higher ability scores (p=0.003) on the EGAD assessment. Caucasian students of both genders were positively correlated with score (r</span><span style = "vertical-align:super;">2</span><span> = 0.51</span><span style = "font-style:italic;">, </span><span>p<0.001, n=444) and ability (r</span><span style = "vertical-align:super;">2</span><span>= 0.47, p<0.001, n=444). Based on the evidence gathered through our analyses, the scores obtained from the EGAD can validly and reliably distinguish between levels of content knowledge on introductory neurophysiology principles for students in introductory plant and physiology courses. 
</span></div></div>

[STEPS]
TITLE: Bulk RNA sequencing
AUTHORS: anita.adami

[DESCRIPTION]
This protocol describes how to perform bulk RNA sequencing

[STEPS]
SECTION: Bulk RNA sequencing
1. Total RNA was isolated from nuclei (Nuclear Extraction from Tissue for FACS), cell culture samples, or tissue using the RNeasy Mini Kit (Qiagen).
SECTION: Bulk RNA sequencing
2. Libraries were generated using Illumina TruSeq Stranded mRNA library prep kit (poly-A selection) and sequenced on a NextSeq500 (PE 2×150bp).
TITLE: Postnatal astrocyte labeling by electroporation (PALE)
AUTHORS: Shiyi Wang

[DESCRIPTION]
Postnatal astrocyte labeling by electroporation (PALE)

[STEPS]
1. **Animal Preparation**
1.1. - Sedate late P0/early P1 mice by hypothermia until anesthetized.
2. **Plasmid Preparation**
2.1. - Prepare plasmid DNA mixed with Fast Green Dye for visualization.
3. **Injection Procedure**
3.1. - Inject 1 μl of plasmid DNA mixture into the lateral ventricle of one hemisphere using a pulled glass pipette (Drummond).
4. **shRNA Knockdown Experiments in Wild-Type CD1 Mice**
4.1. - Prepare 1 μl of DNA containing 1 μg of pGLAST-PBase and 1 μg of pPB-shRNA-mCherryCAAX for injection.
5. **Astrocyte Labeling in WT and LRRK2 G2019Ski/ki Mice**
5.1. - Prepare 1 μl of DNA containing 1 μg of pGLAST-PBase and 1 μg of pPB-mCherry-CAAX for injection per mouse.
6. **PALE-Mediated Overexpression of Phospho-mimetic Ezrin in shRNA Knockdown Experiments**
6.1. - Prepare 1 μl of DNA mixture containing 0.5 μg pGLAST-PBase, 0.5 μg pPB-shRNA-mCherryCAAX, and 1 μg pZac2.1-GfaABC1D-Ezrin T567D-BioID2-HA.
7. **Phospho-Dead Ezrin Overexpression in WT and LRRK2 G2019Ski/ki Mice**
7.1. - Prepare 1 μl of DNA mixture containing 0.5 μg pGLAST-PBase, 0.5 μg pZac2.1-gfaABC1D-mCherry-CAAX, and 1 μg pZac2.1-GfaABC1D-Ezrin T567A-BioID2-HA.
8. **Electrode Placement and Electroporation**
8.1. - Orient electrodes with the positive terminal above the frontal cortex and the negative terminal below the chin of the pups.
8.2. - Apply 5 discrete 50 ms pulses of 100 V spaced 950 ms apart.
9. **Recovery**
9.1. - Recover pups on a heating pad and return them to their home cage.
9.2. - Monitor pups until collection at P21.
10. **Animal Monitoring and Sample Collection**
10.1. - Monitor all animals for health status until collection at P21.
11. **Brain Section Examination**
11.1. - Examine brain sections for the presence of electroporated cells before subsequent staining procedures.
12. Notes:
12.1. - Ensure all procedures are performed in compliance with institutional guidelines for animal care and use.
12.2. - Maintain sterile conditions during plasmid preparation and injection procedures.
12.3. - Optimize electroporation parameters for consistent and reproducible results.
TITLE: Challenge Assay
AUTHORS: Elizabeth Fozo

[STEPS]
?. [Challenge Assay]
Have plenty of BHI plates ready. Calculate the number of plates you will need for dilutions (if you expect more killing for a certain strain/condition/time point, dilutions will be lower), label plates accordingly.
?. [Challenge Assay]
Prepare plenty of tubes for serial dilutions. Make a solution of 0.9% NaCl, and to dilution tubes, add 900µL. Cap and autoclave.
?. [Challenge Assay]
Oftentimes, as time progresses, the challenge will do more damage, killing more cells, meaning that for later time points you may not need as many dilution tubes for serial dilutions, though protective fatty acids may increase survival. This will be something to consider before planning your experiment.
?. [Challenge Assay]
Prepare overnights of strains.
?. [Challenge Assay]
In the morning, dilute cells to an optical density of 0.01 in10 mL supplemented BHI.
?. [Challenge Assay]
Give an appropriate amount of time for them to reach the same OD; if necessary, start cultures in the incubator at varying times depending on the strain/condition.
?. [Challenge Assay]
For long-term supplementation, add a supplement to BHI before adding O/N culture.
?. [Challenge Assay]
For short-term supplementation, grow cells to an OD of ~0.225-0.25 before adding a supplement, then incubate another half-hour before moving on to step 5.
?. [Challenge Assay]
Harvest cells – centrifuge; flood with PBS, wash 2x, resuspend in 10 mL BHI.
?. [Challenge Assay]
Note for daptomycin you need to use calcium for it to be active. So, grow cells, harvest them (with washing steps as required), then resuspend in BHI + 1.5 mM calcium chloride.
?. [Challenge Assay]
Take time point zero. Serially dilute culture in dilution tubes containing 0.9% NaCl.
?. [Challenge Assay]
Add appropriatevolume/concentration ofchallenge(SDS,daptomycin, bile,etc.)
?. [Challenge Assay]
For daptomycin, transfer 2.5 mL of cells into a glass tube, and add an appropriate volume of daptomycin to each (we use15 µg/mL as standard).
?. [Challenge Assay]
At time points 15, 30, 60 minutes: Serially dilute as in step 6 to appropriate dilutions.
?. [Challenge Assay]
Plate dilutions on BHI.
?. [Challenge Assay]
As you go, plate dilutions on BHI plates. It is okay if you are behind on time to let the culture sit in dilution tubes, but don’t squirt dilutions on the plates and allow them to sit for too long as this may cause issues with spreading.
?. [Challenge Assay]
Incubate plates overnight. Count plates in the morning, and calculate the log ratio of survivors.
TITLE: Endosome isolation 
AUTHORS: Chuyu Chen

[DESCRIPTION]
Subcellular fractionation to isolate early and late endosomes (EEs and LEs) by performing a series of centrifugation steps.

[STEPS]
SECTION: Continuous sucrose gradient tube preparation
7. add 2.5 ml 0.45M sucrose on top of frozen buffer S3 and wait for 10 min or until it frozen

SECTION: Continuous sucrose gradient tube preparation
8. keep the frozen tubes at -80 until experiment day
SECTION: Continuous sucrose gradient tube preparation
1. 
prepare 0.5M and 2M sucrose buffer
SECTION: Continuous sucrose gradient tube preparation
2. prepare buffer S2 by mixing 0.5M and 2M sucrose at 1:2 and buffer S3 at 2:1 ratio 
SECTION: Continuous sucrose gradient tube preparation
3. place Beckman ultracentrifuge tube (No.344059) on dry ice
SECTION: Continuous sucrose gradient tube preparation
4. add 2.5ml 2M sucrose in the tube and wait for 10 min or until it frozen.

SECTION: Continuous sucrose gradient tube preparation
5. add 2.5 ml buffer S2 on top of frozen 2M sucrose and wait for 10 min or until it frozen

SECTION: Continuous sucrose gradient tube preparation
6. add 2.5 ml buffer S3 on top of frozen buffer S2 and wait for 10 min or until it frozen

SECTION: Continuous sucrose gradient tube preparation
9. place the frozen tubes at 4C for 300 min or until defrosted before use

SECTION: Fractionation
10. Dissect striatum from mice, fresh freeze the tissue with dry ice
SECTION: Fractionation
11. Prepare homogenization buffer (HB): 150mM Nacl, 10mM HEPES, 1mM EGTA, 0.1M MgCl
SECTION: Fractionation
12. Homogenize striatum tissue with 1ml HB containing 1x Halt protease and phosphatase inhibitor (EDTA-free) by Dounce homogenization on ice (30 strokes)
SECTION: Fractionation
13. Centrifugation 10 min, 3,000g, 4C

SECTION: Fractionation
14. load postnuclear supernatants onto the top of continuous sucrose gradient 
SECTION: Fractionation
15. Centrifugation 1080 min, 200,000g, 4C (Accel and Decel -> slow)

SECTION: Fractionation
16. Collect fractions from top to bottom of the gradient
SECTION: TCA precipitation
17. add equal volume of ice-cold 55% TCA and incubate on ice for 30 min

SECTION: TCA precipitation
18. centrifugation 10 min, 20,000g, 4C

SECTION: TCA precipitation
19. wash pellet with 1ml ice-cold acetone centrifugation 10 min, 20,000g, 4C

SECTION: TCA precipitation
20.   for 2 times. Total 3 washes

SECTION: TCA precipitation
21. resuspend pellet with 70ul of 1% SDS buffer (1% SDS, 10mMTris, 2mMEDTA) containing 1x Halt protease and phosphatase inhibitor (EDTA-free), sonication for 3 s and followed by incubation on orbital shaker for 1hr at room temperature.

SECTION: TCA precipitation
22. Store samples at -80 for western blot assay
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol we have used for the electroporation of mRNA encoding reporter gene Lucipherase into following organisms: <em>Chromera velia</em>, <em>Alexandrium minutum</em>, <em>Euglena gracilis</em>, <em>Pyramimonas parkease</em>, <em>Pyramimonas orientalis</em><em>, Eutreptiella gymnastica, Pseudonitzschia multiseries </em><span style="text-decoration: underline;"></span>and <em>Trichomonas vaginalis </em>(a control). In any case we have not detected a specific lucipharease activity.</p>

[STEPS]
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TITLE: Transcardial Perfusion in Mice for Synaptic Evaluation and QUantification by Imaging Nanostructure (SEQUIN) Analysis
AUTHORS: Saroj Kumar Sah, Thomas Biederer

[DESCRIPTION]
This protocol details the method for transcardial perfusion in mice for Synaptic Evaluation and QUantification by Imaging Nanostructure (SEQUIN) Analysis. This transcardial perfusion can also be used for immunohistochemical analysis of p-Syn staining along with synaptic proteins.

[BEFORE_START]
Before starting perfusion, a lab mat should be placed inside the chemical hood to absorb any spills. An anesthetic drug cocktail should be prepared based on the desired concentration and volume sufficient for the number of mice.

[GUIDELINES]
A record of mouse anesthesia and surgery procedures, drugs used and any leftovers should be maintained, and any leftovers should be stored/disposed of according to the Animal Care and Use Committee (IACUC) and Environmental Health & Safety guidelines.

[STEPS]
SECTION: Transcardial Perfusion
1. Make a mixture of ketamine (100 mg/kg) and xylazine (10 mg/kg), sufficient to anesthetize all mice. 

SECTION: Transcardial Perfusion
2. Administer anesthesia to one mouse via intraperitoneal injection and place it in a clean cage until the anesthesia takes effect. Perform a firm toe pinch to ensure the mouse does not react, confirming the absence of pain sensation before proceeding.
SECTION: Transcardial Perfusion
3. Place the mouse, abdomen up, on a stack of Styrofoam and make it immovable by pinching the toes with a 27-gauge needle through the Styrofoam. Spray the abdomen with 70% ethanol. Using forceps, grasp the skin below the ribcage and cut upward with scissors, cutting through the ribcage to expose the heart.
 

SECTION: Transcardial Perfusion
4. Remove pericardium and peripheral fat to expose heart. Hold back ribs with hemostats. 


SECTION: Transcardial Perfusion
5. Insert a blunted 27G needle tip into the left ventricle and inject ice-cold 1x phosphate-buffered saline (PBS) for4 min, followed by ice-cold 4% paraformaldehyde (PFA) in PBS pH 7.6 for 4 minat a rate of 6 mL/min using a peristaltic pump.
 




SECTION: Transcardial Perfusion
6. After perfusion is complete, carefully remove brain using scissors and transfer to 4% PFA. Store the brain in 4% PFA at 4 °C for 12 to 18 h. Rinse the fixed brains in 1x PBS and cut coronally into 40 to 50 µm-thick slices using a vibratome (Leica VT1000 S) for SEQUIN.


 

SECTION: Perfusion Solutions
7. Weigh PFA under the hood.
SECTION: Perfusion Solutions
8. Heat 1x PBS, transfer PFA into the 1x PBS, and stir (inside chemical hood) to make 4% PFA.
SECTION: Perfusion Solutions
9. Let dissolve the PFA around 60 °C (use the thermometer). Do not let the temperature exceed 65 °C(remove from hot plate temporarily if necessary).


SECTION: Perfusion Solutions
10. Add NaOH to make the PFA dissolved (it should make the PFA clear). Let the PFA cool down to Room temperature . Add HCL to make the pH  pH 7.6. 

SECTION: Perfusion Solutions
11. Filter the 4% PFA into a bottle/w lid.
SECTION: Perfusion Solutions
12. Store the PFA covered at4 °C and use it for perfusion within 12 h.
TITLE: SenNet Case Selection Protocol | JHU TMC
AUTHORS: Kyu Sang Han, Fan Wu, Sashank Reddy, Denis Wirtz, Joel Sunshine, Pei-Hsun Wu

[DESCRIPTION]
Inclusion
Age below 30 or above 50.
English Speaking.
Able to provide consent.
Resected pancreas tissue.
Clear and normal
structured islets of Langerhans, acinus, and ducts from H&E.
 
Exclusion
Large region of squamous
metaplasia or PanIN.
Abnormal or missing hematoxylin
nucleus stain.
Strong personal history of
diabetes or pancreatitis.
Large area percentage of
stromal cells.

[STEPS]
SECTION: Disclaimer
1. Patient samples will be obtained from distal pancreatectomy, splenectomy, and cholecystectomy. No other procedures or follow-up is needed for research purposes.
SECTION: Inclusion
2. Age below 30 or above 50.
English Speaking.
Able to provide consent.
Resected pancreas tissue.
Clear and normal structured islets of Langerhans, acinus, and ducts from
H&E.
SECTION: Exclusion
3. Large region of squamous metaplasia or PanIN.
Abnormal or missing hematoxylin nucleus stain.
Strong personal history of diabetes or pancreatitis.
Large area percentage of stromal cells.
TITLE: Spectrophotometric Quantification of Betacyanins in Plant Tissue Produced from the RUBY Reporter Gene
AUTHORS: matthew.morgan

[DESCRIPTION]
RUBY is a visual reporter gene that produces beatnin, a betacyanin that appears red to violet in colour (He et al., 2020). To assay betanin (and other betalains) a spectrophotometric approach has been employed, as seen in Stintzing et al. (2003) and Grützner et al. (2021).

[STEPS]
SECTION: Sample Preparation
3. Take 20-50 mg of frozen tissue and add to 2 ml tubes. Keep on ice.
SECTION: Sample Preparation
4. Grind down tissue using plastic micro-pestle and add methanol buffer (50% methanol, 1 mM ascorbic acid, 0.5% formic acid) at 10% w/v - i.e. for 35 mg of tissue add 350 ul methanol buffer.
SECTION: Sample Preparation
5. Dilute samples with mili-Q so that absorption values fall within 0.8 - 1.0 (as previously calculated by Stintzing et al. 2003). Grützner et al. (2021) found that a 12 fold dilution performed well but this could differ.

SECTION: Spectrophotometric Quantifications
6. Put cuvettes or 96 well plates into spectophotometer and read absorbtion at 538 nm (Stintzing et al. 2003).
SECTION: Spectrophotometric Quantifications
7. Using the formula BC = (A * DF * MW * 1000) / ( ε x L) calculate the concentration of betacyanins.

BC = betanin content (mg/L)    A = absorbance (538 nm)    DF = dilution factor    MW = molecular weight (550.47 g/mol)    L = cuvette path length    ε =   molar extinction coefficient (60, 000 L/(mool cm)


SECTION: Sample collection
1. Punch out leaf discs (as many as possible in the target area) of areas expressing betanin.
SECTION: Sample collection
2. Put leaf discs in 2 ml micro-centrifuge tubes and snap freeze in liquid nitrogen then store in -80 freezer until required.
TITLE: Differentiation of hPSCs into Dopamine neurons
AUTHORS: Beatrice Weykopf

[DESCRIPTION]
This protocol describes the differentiation of hPSCs into dopaminergic neurons modified from Kriks et al., 2011 and Ryan et al., 2013

[STEPS]
1. Day -1: seed 200K cell/cm2 in mTeSR+ medium supplemented with 10µM Rock inhibitor (RI) onto Martigel coated TC plates
2. Day 0: Differentiation initiation. Aspirate mTeSR+ medium and add SRM media supplemented with 200nM LDN193189 + 10µM SB431542
3. Day 1: SRM + LDN + SB supplemented with 100 ng/ml FGF8b, 100 ng/ml ShhC25II and 2µM Purmorphamine
4. Day 2: SRM + LDN + SB+ FGF8b + ShhC25II + Pur
5. Day 3: SRM + LDN + SB+ FGF8b + ShhC25II + Pur +3 µM CHIR99021
6. Day 4: no feed if medium is not consumed
7. Day 5: 75% SRM + 25% Neurobasal + LDN + SB+ FGF8b + ShhC25II + Pur +CHIR
8. Day 6: no feed if medium is not consumed
9. Day 7: 50% SRM + 50% Neurobasal + LDN + CHIR
10. Day 8: no feed if medium is not consumed
11. Day 9: 25% SRM + 75% Neurobasal + LDN + CHIR
12. Day 10: no feed if medium is not consumed
13. Day 11: 100% Dopa mat basal medium + CHIR
14. Day 12: no feed
15. Day 13: Passage cells in a ratio of 1:1 onto matrigel-coated dishes with 30-60min Accutase treatment. Spin down the cells in Dopa mat basal and resuspend in Dopa mat medium supplemented with 10µM Y27632 and CHIR.
16. Day 20-24: Cells are plated on Poly-D-lysine and laminin coated plates in a density of 2-2.5M cells / 6 well. Cells are dissociated using 60-90min Accutase supplemented with 10µM Y27632.
17. Day 25-27: 3µM cytosine arabinoside is added to Dopa mat media. Day 27 cells are washed twice with Dopa mat basal medium to remove any cytosine arabinoside residues. 

18. Around Day 30: Cells are dissociated using Accutase supplemented with 10µM Y27632 and plated in onto Poly-D-lysine and laminin coated dishes. The final density depends on the assay.
TITLE: Electroporation of Caecitellus sp. with FITC-dextran
AUTHORS: Nick Irwin, Elisabeth Hehenberger, Patrick Keeling

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Caecitellus </span><span>sp.</span><span style = "font-weight:bold;"> </span><span>is a small phagotroph belonging to the biocosoecid order of the stramenopiles. </span><span style = "font-style:italic;">Caecitellus </span><span>is found across global oceans but it's role in marine ecosystems, as with many heterotrophic flagellates, is unclear. </span></div><div class = "text-block"><span>We isolated a strain of </span><span style = "font-style:italic;">Caecitellus</span><span> (small subunit rRNA gene was 99.53% identical to </span><span style = "font-style:italic;">Caecitellus parvulus </span><span>and </span><span style = "font-style:italic;">Caecitellus paraparvulus</span><span>) from a contaminated culture of </span><span style = "font-style:italic;">Oxyrrhis marina</span><span> and assessed its amenability to transformation. </span></div><div class = "text-block"><span>Fluorescein isothiocyanate conjugated to dextran was successfully introduced into </span><span style = "font-style:italic;">Caecitellus </span><span>sp. using a high voltage exponential decay pulse (1000 V, 10 µF, ∞ Ω) from a BioRad GenePulser Xcell (cuvette width 0.2 cm). Cell viability was confirmed by observing motility and fluorescence 24 h after electroporation (Figure 1). Lower voltage pulses did not result in FITC uptake. Estimates of transformation effifiency were difficult to obtain due to the small size of the cells.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bakkaw.jpg" /></div><div class = "text-block"><span>Unfortunately, the isolated strain of </span><span style = "font-style:italic;">Caecitellus</span><span> perished in an incubator malfunction. However, these results may be transferable to </span><span style = "font-style:italic;">Caecitellus parvulus</span><span>, a model for heterotrophic flagellates. 
 </span></div></div>

[STEPS]
?. [Culturing]
Cells were grown in f/2 media in a 20 C incubator on a 12:12 light:dark cycle.Cells fed on natural populations of bacteria in the culture and did not need supplemental food.
?. [Electroporation]
Spin down cells at 3000 x g for 10 minutes.Resuspend cells in 100 uL of BioRad Gene Pulser electroporation buffer with 2 mg/mL FITC-dextran.Electroporate the cells using a high voltage exponential decay pulse (1000 V, 10 µF, ∞ Ω) from a BioRad GenePulser Xcell (cuvette width 0.2 cm). After electroporation, add 900 uL of f/2 and transfer to a 1.5 mL centrifuge tube.Wash the cells once by spinning at 7000 xg for 3 minutes, removing the supernatant, and resuspending in 1 mL fresh f/2Place cells in a 24-well plate and allow them to recover at room temperature in the dark for 24 hours
?. [Observations]
Observe the cells 24 hours after electroporation.Successful transformants will appear strongly fluorescent green and will be swimming.
TITLE: Husbandry guidelines for the safe brumation of two lacertid lizard species (Iberolacerta monticola and Podarcis lusitanicus) in laboratory conditions
AUTHORS: Frederico M Barroso

[DESCRIPTION]
Lacertid lizards are often used for a range of laboratory studies in reptile behaviour, physiology, ecology etc. However, there is a distinct lack of primary information regarding the husbandry of these animals under captive, laboratory conditions. This lack of information is even more apparent when looking for standard husbandry best practices for the brumation of small lizards in a captive setting. Hence, when faced with the need to overwinter captive populations of two lacertid lizard species from a montane environment (Iberolacerta monticola and Podarcis lusitanicus), the need arose to establish a conservative protocol that would allow the animals to safely go through their seasonal activity cycle (and brumate) in a controlled laboratory environment. This protocol therefore addresses that gap as it aimed to simulate a simplification of the conditions thought to be experienced by these animals while brumating. Ultimately, through this protocol, these species were successfully overwintered in a laboratory setting without any casualties (during the brumation period) and with no significant effect on the body condition of the animals. Hence, this protocol hopes to provide a baseline framework, on which other studies may capitalise, build upon or adjust, for the safe brumation of lizards in a laboratory setting. 

[STEPS]