eamag/protocols-with-steps huggingface dataset:
TITLE: GFP-VSV Infection
AUTHORS: Christopher Rousso, Alison Macdonald

[DESCRIPTION]
GFP-VSV Infection

[STEPS]
1. If infecting transfected cells, infect them 24 hours after transfection. 
2. Remove cells from 37 °C incubator.
3. Replace media with fresh media (with FBS) prior to infection with GFP-VSV.
5. Dilute VSV-GFP in serum-free culture media. Perform the necessary calculations to determine the dilution that is required to obtain the desired MOI (multiplicity of infection).
Note: MOI is the number of viruses/number of cells. For example, a MOI of 1 = 500,000 PFU (plaque forming units) of virus per 500,000 cells. 
Note: We typically start with a dilution range of MOI = 0.01, 0.1, 1 for optimization
6. Infect cells by dropping the diluted virus directly into the wells making sure to disperse the virus around the well. 
7. Gently swirl the plate to help disperse the virus. 
8. Leave the plate for approximately 24 hours in a 37°C CO2 incubator. Then proceed with endpoint analysis.
SECTION: Appendix - Sample Calculation
9. Sample calculation: 
(MO1 = 1; Virus titre = 8.4x108 virus/mL ; 500,000 cells/well;)
Step 1: calculate amount of PFU per well

 

Step 2: calculate how much to dilute the viral stock 
C1V1=C2V2
Where V1 = volume/well you have 
C1 = virus/mL calculated in step 1
C2 = virus titre (stock concentration)
V2 = x (unknown volume to be calculated)
You will add x mL of your virus stock per well. This volume will be low, and thus must be diluted in media for easier pipetting.
4. Thaw VSV-GFP stock aliquot on ice
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em>The MELD Project is an international collaboration aiming to create open-access, robust and generalisable tools for FCD detection. To this end, we will train a neural network classifier on MRI features from FCD patients from multiple centres worldwide.</em></p>
<p><strong> </strong></p>
<p><strong>Protocol 4 provides instructions on how to create lesion masks in FreeSurfer using tkmedit.</strong></p>

[BEFORE_START]
<p><strong>Lesion masks should ideally be created for all patients included in the MELD Project. </strong></p>
<p> </p>
<p>If the patient has a radiological diagnosis of FCD, the lesion mask should be done using the 3D T1 scan and 3D FLAIR (where available). If 3D FLAIR is not available, 2D FLAIR may be of help in defining the lesion. You may need assistance from a neuroradiologist at your centre for the subtle lesions.</p>
<p> </p>
<p>If the patient is MRI-negative but with histological confirmation, the post-operative scan and other corroborative evidence such as EEG, PET, sEEG etc. can be used to help define where the lesion was on the preoperative scan in order to create the lesion mask.</p>
<p> </p>
<p>If it is NOT possible to create a lesion mask. Please still include this patient in the study. In the csv file named <em>MELD_[site code]_participants.csv </em>, in the lesion mask column mark as 0.</p>
<p> </p>
<p><strong>Radiological features of FCD:</strong></p>
<ul>
<li>Abnormal cortical thickness</li>
<li>Blurring of the grey-white matter boundary</li>
<li>Increased signal intensity of FLAIR / T2 (transmantle sign in FCD IIB)</li>
<li>Abnormal folding pattern</li>
<li>Hemispheric asymmetry</li>
</ul>
<p> <img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/tv2ja5e.png" data-src="https://s3.amazonaws.com/pr-journal/tv2ja5e.png" data-ofn="Radiological.png" /></p>
<p><strong> </strong></p>
<p><strong>The Steps detail how to create a lesion mask in FreeSurfer using the viewer <em>tkmedit </em>, how to move the lesion mask to the FreeSurfer surfaces and save as a .mgh surface file. </strong></p>
<p><strong> </strong></p>
<p><strong>If your centre has an established method to create the lesion masks or have already created lesion masks, you do not need to redo them or change your method. The lesion masks will need to be registered to the FreeSurfer surfaces. Please see <em>Supplementary Steps </em>for how to register .nii lesion masks to FreeSurfer surfaces. </strong></p>
<p><strong> </strong></p>
<p><strong>If you have any questions or run into problems, please feel free to contact the MELD project: (</strong><a href="mailto:meld.study@gmail.com" target="_blank"><strong>meld.study@gmail.com</strong></a><strong>).</strong></p>
<p><strong> </strong></p>
<p><strong> </strong></p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A defined artificial seawater medium for growing bacteria.</p>

[BEFORE_START]
<p>Acid wash all glassware with 10% HCl and rinse well with dH<sub>2</sub>O.</p>

[GUIDELINES]
<p>Increase or decrease amount proportionally to prepare stock quantities as needed. Add dry reagents slowly to allow for dissolution in between each addition. Solutions will be prepared and autoclaved separately, except for carbon and vitamins which are filter sterilized through 0.2 micron PES membranes. Each autoclaved solution should be cooled completely before combining - remember to autoclave extra bottles or graduated cylinders as needed for measuring and mixing. The carbon source should only be added after autoclaved solutions have cooled; it is filter sterilized into the medium and can be added during mixing or at any time before setting up a culture. Extra solutions can be stored frozen or at 4 Celsius. This recipe is for preparing 1 L of liquid medium, but plates can be made as well with slight variation.</p>

[STEPS]
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TITLE: WT-1 Staining Protocol for Podocytes
AUTHORS: Frank Brosius

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This protocol describes a protocol used by some DiaComp members to detect podocyte nuclei in rodent glomeruli.</div><div class = "text-block"><span style = "font-weight:bold;">Edited by</span><span>: Brosius Laboratory</span></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/bakkun.jpg" /></div></div>

[STEPS]
?. Day 1. Deparaffinize/hydrate1. Dip in Xylene 5 minutes 2x (carry out in the hood)
2. Dip in 100% ETOH 5 minutes 2X
3. Dip in 95% ETOH 5 minutes once
4. Dip in 70% ETOH 5 minutes in once
5. Dip in dH²0 5 minutes 2X
6. Dip in PBS 5 minutes once7. Place the slides in jar with preheated Retrieve All 1 and incubate 2 hrs in 90ºC water bath
8. Remove and let it cool down for 5-10 minutes
9. Pour off solution (can be reused 5-10 times) and add dH²O10. Rinse slides in two change of dH²O 4-5 minutes each 11. Soak in PBS and store at 4ºC.
?. Day 2. Immunoperoxidase1. Blot dry slide (leave the section wet)
2. Incubate 20 minutes at room temperature (RT) with blocking serum (Follow directions of Vectastain ABS Kit for dilution).
3. Dilute WT-1 antibody (first antibody): 1:200 dilution in 1%BSA. Blot excess serum and incubate with first antibody for 2 hours at RT (2ug/section in 100ul).4. Wash in PBS 5-10 minutes (2x changes) and blot dry (don’t touch the section)
5. Incubate with second antibody for 1 hour (Follow directions of Vectastain ABS Kit for dilution)6. Wash in PBS 5-10 minutes (2x changes)
7. Quench endogenous peroxidase by dipping in 1% H²O² inmethanol for 45 minutes
8. Wash in PBS 10-15 minutes (2x changes)
9. Incubate with ABC Reagent for 1 hour (Follow direction ofVectastain ABS Kit for dilution).10. Wash in PBS 5-10 minutes and prepare DAB Tablets (dissolve 1 set in 5 ml ddH²O and filter it)
11. Staining: add 100ul or so DAB substrate to cover the whole section and check under the microscope for podocyte nuclei (~1 minute).12. Stop reaction in dH²O13. Dip in dH²O for 5 minutes 2x14. Dip in 70% ETOH 5 minutes once15. Dip in 95% ETOH 5 minutes once16. Dip in 100% ETOH 5 minutes 2X17. Dip in Xylene 5 minutes 2x (carry out in the hood)18. Mount the slides with permount.
TITLE: seqFISH Hyb Station
AUTHORS: Long Cai, Linus Eng, Nico Pierson, Chris Cronin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The seqFISH Hyb Station is a custom built platform consists of a liquid handling pump , a fluidic switcher , and a 96-well plate auto-sampler which aids seqFISH experiments by flowing hybridization reagents and washes in and out of a flow cell containing the biological sample. Researchers set up multi-day experiments to perform chemistries such as hybridizations, washes, and stripping. In each round of hybridization, the auto sampler handle will move to the designated well and approximately 100uL of readouts reagents are flowed into the custom made flow cell to allow hybridization. Following by imaging with a fluorescent microscope, the signals are extinguished by flowing in excess high formamide solution to melt away the readout probes. These steps are repeated until all hybridization rounds are completed. The controls of seqFISH Hyb Station and imaging are controlled and integrated using the open source software Micro-Manager.</div></div>

[STEPS]
TITLE: Microfluidic device platform operation for processing tissue into single cells 
AUTHORS: Jeremy Lombardo

[DESCRIPTION]
Protocol for operating Minced Digestion and Integrated Dissociation and Filtration Devices

[STEPS]
SECTION: Minced Digestion Device Operation
1. Affix 0.05” ID tubing (Saint-Gobain, Malvern, PA) to Minced Digestion Device inlet and outlet hose barbs.
SECTION: Minced Digestion Device Operation
2. Connect tubing to an Ismatec peristaltic pump (Cole-Parmer, Werheim, Germany) with 2.62 mm ID tubing (Saint-Gobain, Malvern, PA).
SECTION: Minced Digestion Device Operation
3. Incubate devices and tubing with SuperBlock (PBS) blocking buffer (Thermo Fisher Scientific, Waltham, MA) at room temperature for 15 min to reduce non-specific binding of cells to channel walls.
SECTION: Minced Digestion Device Operation
4. Flush out SuperBlock with PBS+1% BSA (PBS+).
SECTION: Minced Digestion Device Operation
5. Load minced tissues into the device tissue chamber through the luer inlet port.
SECTION: Minced Digestion Device Operation
6. Prime device and tubing with 0.25% collagenase type I solution (StemCell Technologies, Vancouver, BC), and close off the luer port using a stopcock.
SECTION: Minced Digestion Device Operation
7. Place the experimental setup (device, tubing, and peristaltic pump) inside a 37°C incubator to maintain optimal enzymatic activity.
SECTION: Minced Digestion Device Operation
8. Recirculate the collagenase solution through the device and tubing using the peristaltic pump at a flow rate of 10 or 20 mL/min for the desired length of time.
SECTION: Integrated Dissociation/Filter Device Operation
9. Following processing with the minced digestion device, connect 0.05” ID tubing (Saint-Gobain, Malvern, PA) from the outlet of the minced digestion device to the inlet of the integrated dissociation and filtration device. If recirculation is intended, connect tubing from the cross-flow outlet to the peristaltic pump, while the outlet of the integrated device is closed off with a stopcock.
SECTION: Integrated Dissociation/Filter Device Operation
10. Pump fluid through the dissociation component at 10 mL/min flow rate. For final collection of the sample, close off the cross-flow outlet with a stopcock, and pump sample through at 10 mL/min and collect from the effluent outlet.
SECTION: Integrated Dissociation/Filter Device Operation
11. Following all experiments, flush out devices with 2 mL PBS+ to collect any remaining cells. For time interval recovery, follow each PBS+ wash with repriming of the device and tubing with collagenase solution, and reconnect the outlet of the minced digestion device to the peristaltic pump for continued recirculation until the next collection period.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Reagent List: </strong><br />Cell Staining Buffer (BioLegend Cat. No. 420201) <br />Red Cell Lysis Buffer (BioLegend Cat. No. 420301) <br />7-AAD Viability Staining Solution (BioLegend Cat. No. 420403) <br />TruStain FcX™ (anti-CD16/32, BioLegend Cat. No. 101319) <br />Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301)<br /><br /><strong>References:<br /></strong>Current Protocols in Cytometry (John Wiley &amp; Sons, New York), Unit 6 Phenotypic Analysis.

[STEPS]
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TITLE: P1 transduction
AUTHORS: Elizabeth Fozo

[STEPS]
?. [Prepare P1 phage]
Inoculate a single colony into 5 ml LB medium (+ appropriate antibiotic if needed)
?. [Prepare P1 phage]
Shake at   overnight
37 °C
?. [Prepare P1 phage]
Dilute to be 0.01 in 10 ml LB medium  +   of 1 M CaCl2 + 0.2% glucose final
50 µl
?. [Prepare P1 phage]
Grow at   for 60 minutes (don’t want the cells to get much above 0.1 OD600)
37 °C
?. [Prepare P1 phage]
Add   ,  , or   P1KC phage lysate
10 µl
50 µl
100 µl
?. [Prepare P1 phage]
Grow at   until lysed (ideally~3 hr)
37 °C
?. [Prepare P1 phage]
Add   drops chloroform, vortex for 30 seconds
200 µl
?. [Prepare P1 phage]
Spin at ? for 10 minutes
?. [Prepare P1 phage]
Transfer the supernatant into a new 15 ml tube
?. [Prepare P1 phage]
Add 100 ul chloroform
?. [Prepare P1 phage]
Store in fridge
?. [P1 transduction]
Inoculate a single colony into 5 ml LB medium (+ appropriate antibiotic, if needed)
?. [P1 transduction]
Shake at  overnight
37 °C
?. [P1 transduction]
Spin down cells
?. [P1 transduction]
Resuspend in 5 ml MC buffer (10 mM MgCl2, 5 mM CaCl2)
?. [P1 transduction]
Set the transduction mix in a 1.5 ml Eppendorf tubeTube#1    |    Cells     |   P1 lysate1                    0.1 ml             _2                    0.1 ml           10 ul3                    0.1 ml           50 ul4                    0.1 ml           100 ul5                         -                100 ul
?. [P1 transduction]
Incubate at RT for 20 min without shaking
?. [P1 transduction]
Add  1 M NaCritrate
20 µl
?. [P1 transduction]
Shake at   for 1 hr
37 °C
?. [P1 transduction]
Plate   on LB plate + appropriate antibiotic
100 µl
?. [P1 transduction]
Incubate plate at   overnight
37 °C
?. [P1 transduction]
Examine colonies = transductants
TITLE: Obtain LB agar plates (with antibiotics)
AUTHORS: An.Huang

[DESCRIPTION]
LB plate is a fundamental laboratory consumable. This protocol helps make basic LB agar plates.

[STEPS]
1. Weigh 40 g LB nutrient agar powder. 

 

2. Dissolve the powder in 1000 mL distilled water.
 


3. Autoclave the melting LB agar at 121 °C for 15 min.
 

5. (Optional) If the plate is required to contain a certain antibiotic, add a certain amount of antibiotics.
Here ampicillin is used as an example:
Add 1 mL 1000X ampicillin stock solution into 1000 mL melting LB agar. Mix well.
 

 


4. When autoclaving procedure is finished, wait until the flask cools to around 40 °C, which will not hurt the palm.
 


6. Pour melting agar into empty petri dishes until the melting agar has completely cover the bottom of the petri dishes. Avoid bubbles.
 

 

7. Leave the plates to cool and the lids ajar.
8. Wait until all agar plates solidified. Invert the plates and store at 4 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Dairy cows affected with subclinical mastitis can be sources of virulent, antimicrobial-resistant Staphylococci to humans by the excretion of the bacteria through their milk. This study focussed on the phenotypic and genotypic antibiotic resistance patterns of Staphylococci isolated from dairy cows during the early dry period.  Among 96 isolates of Gram positive cocci from 157 cows, 76 were identified as Coagulase Negative Staphylococci and the remaining twenty were <em>Staphylococcus aureus.</em> Typical amplicons of coagulase gene were obtained for all twenty samples of <em>S. aureus</em> with three major coagulase types being identified as giving 627 bp (40 %), 910 bp (35 %) and 710 bp (25 %) long PCR products. The <em>groEL</em> gene was amplified in PCR of all 76 isolates of Coagulase Negative Staphylococci, and incubation of PCR products with restriction enzyme PvuII yielded three distinct PCR-RFLP fragment patterns bearing resemblance to <em>S. chromogenes </em>and<em> S. hyicus. </em>Highest sensitivity of Coagulase Negative Staphylococci was noted for Azithromycin (92.5%) and the least to Tetracyclines (76.3%), whereas for <em>S. aureus,</em> it was Cefoperazone (95%) and Azithromycin (72.2%) respectively. Phenotypic resistance to Oxacillin (25 isolates), and Cefoxitin (11 isolates) was detected by dilution method with a commercial strip (Ezy MIC<sup>TM</sup>).  Genotypic resistance to b-Lactam antibiotics was found in 65 (34 with <em>mecA</em> gene and 31 with <em>blaZ</em> gene) isolates. Eighteen isolates possessed both the genes, with the PVL gene for virulence being detected in five of them. Nine isolates which had <em>mecA</em> gene were phenotypically susceptible to oxacillin while phenotypic resistance to oxacillin was observed in seven isolates that did not have either <em>mec</em>A or <em>blaZ</em> gene. This is the first report of persistent Staphylococcal infections possessing PVL gene and high level of genotypic resistance to b-Lactam antibiotics in small- holder dairy cattle from India.  </p>

[STEPS]
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TITLE: Bead preparation protocol
AUTHORS: Peter H L Krijger, Tim A Hoek, Sanne Boersma, Lieke I P M Donders, Maaike M C Broeders, Mark Pieterse, Pim W Toonen, Ive Logister, Bram M P Verhagen, Marjon J A M Verstegen, Thomas W van Ravesteyn, Rene J T M Roymans, Francesca Mattiroli, Jo Vandesompele, Monique Nijhuis, Stefan Meijer, Anton van Weert, Edwin Dekker, Fred J Dom, Rob Ruijtenbeek, Lieven B J van der Velden, Jeroen H B van de Bovenkamp, Martijn Bosch, Wouter de Laat, Marvin E Tanenbaum

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">STRIP is a start-to-end streamlined and automated procedure for COVID-19 testing, centering on a single Tecan Fluent liquid-handling robot that can process over 14,000 samples per day. </div><br/><div class = "text-block">Here we describe the protocol to prepare the magnetic beads that are currently used in STRIP. Note, in this protocol we use Sera-Mag SpeedBead Carboxylate-Modified Magnetic Particles (Hydrophobic) from Cytivia, but other beads such as    SeraSil Mag 400 beads (Cytiva) can be used as substitutes. </div></div>

[STEPS]
?. [Bead preparation]
Mix the beads by vortexing for .
?. [Bead preparation]
Put the beads on a rollerbank for .
?. [Bead preparation]
Aliquot 10 ml beads in 50ml tubes.
?. [Bead preparation]
Put the 50 ml tube on the magnet.
?. [Bead preparation]
When the solution is clear carefully remove the supernatant.
?. [Bead preparation]
Add 20 ml 2-Propanol.
?. [Bead preparation]
Mix by vortexing.
?. [Bead preparation]
Repeat step 4-7.
?. [Bead preparation]
Resuspend beads in 10 ml 2-Propanol. Beads resuspended in 2-Propanol can be stored at 4°C for at least 5 days.
?. [Final dilution]
When ready to be used mix by vortexing and transfer the 10 ml beads to 150 ml 2-Propanol.
?. [Final dilution]
Add 20 ml 2-Propanol to the 50 ml tube to resuspend the remaining beads and transfer the diluted beads.
?. [Final dilution]
Repeat step 11.
?. [Bead preparation]
Repeat step 4-5.
?. [Final dilution]
Transfer the diluted beads (Total volume 200 ml) into the spinvessel. Start the spinvessel, this will keep the beads in solution. Beads can be used for 5 days at RT.
TITLE: Morphotyping Fungal Cultures
AUTHORS: Craig Bateman, Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols describes the process of designating fungal morphotypes.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: Adeno-associated virus (AAV) production and administration
AUTHORS: Shiyi Wang

[DESCRIPTION]
Adeno-associated virus (AAV) production and administration

[STEPS]
1. **Transfection of HEK293T Cells**
1.1. - Transfect HEK293T cells with the following plasmids: pAd-DELTA F6, serotype plasmid AAV PHP.eB, and AAV plasmid (pZac2.1-GfaABC1D-Ezrin-BioID2-HA or pZac2.1-GfaABC1D-Ezrin T567A-BioID2-HA).
2. **Cell Lysis and AAV Purification**
2.1. - Three days after transfection, collect cells in a lysis buffer (15 mM NaCl, 5 mM Tris-HCl, pH 8.5).
2.2. - Perform repeat freeze-thaw cycles followed by treatment with Benzonase at 37°C for 30 minutes.
2.3. - Centrifuge lysed cells to pellet debris, and collect the supernatant containing AAVs.
3. **Optiprep Density Gradient Centrifugation**
3.1. - Apply the supernatant containing AAVs to an Optiprep density gradient (15%, 25%, 40%, and 60% iodixanol).
3.2. - Centrifuge at 67,000 rpm using a Beckman Ti-70 rotor for 1 hour.
3.3. - Collect the AAV-enriched fraction from between the 40% and 60% iodixanol layers.
4. **AAV Concentration**
4.1. - Concentrate the AAV fraction by repeated washes with sterile PBS using an Amicon Ultra-15 filtration unit (100 kDa NMWL) to a final volume of approximately 100 μl.
4.2. - Aliquot the concentrated AAVs for storage at −80°C.
5. **Mouse Anesthesia and Surgery**
5.1. - Anesthetize 9-week-old WT or LRRK2 G2019Ski/ki mice using 1.5% isoflurane gas in a stereotaxic frame.
6. **Intravenous AAV Injection**
6.1. - Inject 10 μl of purified AAVs (titer of ~1 x 10^12 GC/ml) into the mouse brain intravenously by injection into the retro-orbital sinus.
7. **Post-Injection Care and Tissue Preparation**
7.1. - Allow mice to recover for 3 weeks until they reach 12 weeks of age.
7.2. - Anesthetize mice with 200 mg/kg Tribromoethanol (Avertin) and perform transcardial perfusion with TBS/Heparin followed by 4% paraformaldehyde (PFA) at room temperature (RT).
8. **Brain Processing**
8.1. - Post-fix harvested brains overnight in 4% PFA, then cryoprotect in 30% sucrose.
8.2. - Embed brain blocks in O.C.T. (TissueTek) and store at −80°C.
9. **Cryosectioning**
9.1. - Cut 30 µm thick brain sections using a Leica CM3050S vibratome.
9.2. - Store sections in a mixture of TBS and glycerol at −20°C for subsequent antibody staining procedures.
10. Notes:
10.1. - Ensure all procedures are performed in accordance with institutional guidelines for animal care and use.
10.2. - Maintain sterile conditions during AAV preparation and injection procedures to prevent contamination.
10.3. - Document all experimental details including AAV titers, injection volumes, and storage conditions for reproducibility.