eamag/protocols-with-steps huggingface dataset:
TITLE: Immunoblots
AUTHORS: wusj, Nancy C. Hernandez Villegas, Iona Thomas-Wright, Richard Wade-Martins, schekman

[DESCRIPTION]
This protocol describes a standard procedure for protein separation and identification of protein of interest. 

Protocol overview:

The steps of this protocol is similar for different sources of protein such as cell lysate, cytosol, or membrane samples, and EV samples. However, there are some specific changes depending on protein of interest, endogenous alpha-synuclein, or type of cells, hiPSC dopamine neurons.

[STEPS]
SECTION: Immunoblots
1. Cell lysate, cytosol, or membrane samples, and EV samples were mixed with SDS sample loading
buffer. For cell lysate, cytosol or membrane samples, 20 μg proteins were loaded. For EV samples, the maximal amount up to 20uL were loaded.
SECTION: Immunoblots
2. Samples with DNAJC5 and α-syn were heated at 55°C for 10 min to prevent aggregation. Other samples were heated at 95°C for 5 min and separated on SDS-PAGE gels (Novex wedgewell 4%-20% Tris-Glycine mini gels, 200 V for 45 min). 
SECTION: Immunoblots
3. Proteins were transferred to PVDF membranes (EMD Millipore, Darmstadt, Germany) in cold room at 0.6 A for 2 h.
SECTION: Immunoblots
4. Blocked membrane with 5% bovine serum albumin in TBST (20 mM Tris pH 7.4, 150 mM NaCl, and 0.1% Tween-20) at Room temperature for 1 h with constant agitation. 60 min 

SECTION: Immunoblots
5. Incubated membranes with primary antibodies at 4 °C overnight.


SECTION: Immunoblots
6. For immunoblots from hiPSC dopamine neurons, samples in loading buffer were heated to 70 °C for 10 min and blocking was carried out with 5% skimmed milk. 

SECTION: Immunoblots
7. For immunoblots of endogenous α-syn in SH-SY5Y cells, PVDF membranes were fixed with 0.4% paraformaldehyde in TBST at room temperature for 30 min  
SECTION: Immunoblots
8. Blots were washed with TBST for 5 times, each time with 5 min agitation. 

SECTION: Immunoblots
9. Incubate membranes with secondary antibodies, anti-rabbit or anti-mouse for 60 min at Room temperature 
 
SECTION: Immunoblots
10. Detection was performed with Supersignal Chemiluminescent substrate and quantified with Fiji/ImageJ.
TITLE: Graphing area from an image series using ImageJ: a simple method
AUTHORS: Vanessa R Ho, Sally P Leys

[DESCRIPTION]
This protocol provides a quick way to quantify change in area of an object in a large series of images, by pixel contrast. It's equivalent to rootpainter, except much simpler, but it does rely on good contrast of light and dark in the region you wish to measure.
Others have used ImageJ for this too, but the method and workflow we provide has been tested successfully by many students and therefore offers a more consistent, standardized process.

[BEFORE_START]
Make sure you have ImageJ downloaded, available here: https://imagej.net/software/fiji/

[STEPS]
1. Create a workflow (macro) in ImageJ with the following steps: This will be run on all your images.
Open ImageJ
Opening an image, 
Selecting Plugin>Macro>Record, which opens up a ‘recorder’ window.
 



2. Recording the macro for image processing: 
Set image type to 8-bit grayscale
Select the rectangular selection tool and draw a rectangle over
the image to create a ROI,
Crop the image to that region >Image>Crop, 
Threshold the image >Image>Adjust>Threshold>Auto>Set>OK, 
Measure the area fraction, >Analyze>Set Measurements>, check the
Area Fraction box (no other measure is needed), >Analyze>Measure. This action sends the measurement to a results window. 
Save the results to a folder by choosing >File>Save as>[choose your folder and type the file name].
Stop the macro recording in the ‘recorder’ window by clicking >Create. 

A Macro.ijm window opens with the macro, click >File>Save as>[choose a
folder and file name for the macro].
Close the image and result window now.
 


 



3. Run the macro on a set of images: 
In the main ImageJ toolbar choose >Process>Batch>Macro, to open the batch process window.
Select the folder which contains the image set to be analysed, and the folder
where the data and the processed images will be placed. 
Copy and paste the macro.ijm file from above into the window, and click >Process. 

The image set will be analysed and the area fraction for each image will be calculated and placed in
a .csv file, which can be opened for graphing or further analysis in Excel or other programs
TITLE: 100ml 5M Sodium Chloride (NaCl)
AUTHORS: Menglin WANG

[DESCRIPTION]
This is a recipe for how to make 100ml 5M Sodium Chloride (NaCl).

[STEPS]
SECTION: 5M Sodium Chloride (NaCl,100ml)
1. Weigh out an amount of 29.22 g of    in an appropriate-size beaker or conical flask (1L volume). Add   80 mL of   .

Precaution: Do not add 100 ml of deionized/Milli-Q water. In most cases, solution volume increases when a large amount of solute is dissolved in a solvent.
SECTION: 5M Sodium Chloride (NaCl,100ml)
2. Dissolve   using a magnetic stirrer or manual shaking with a glass stirring rod.

Tip: You can heat the solution to 60 °C  to accelerate the dissolution process.
SECTION: 5M Sodium Chloride (NaCl,100ml)
3. Transfer the solution to a measuring cylinder or a volumetric flask and adjust the volume to 100 mL with  . Remix it.
SECTION: 5M Sodium Chloride (NaCl,100ml)
4. Transfer the solution to an autoclavable bottle and sterilize it by autoclaving 20 min  at 15 lb/sq.in. (psi) from 121-124 °C  on liquid cycle.
SECTION: 5M Sodium Chloride (NaCl,100ml)
5. Storage: Store the solution at Room temperature.

Tip: NaCl may precipitate with time. Dissolve it by shaking the bottle vigorously.
TITLE: Detection of respiratory viruses or housekeeping genes by qPCR (Taqman) – primers and probes
AUTHORS: José Luiz-Proença Modena, Flavia Escremim de Paula, Eurico Arruda

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Detection of hRSV-A/B; hMPV-A/B (Duplex format of qPCR-Taqman). For final volume reaction of 15µl, add 3µL cDNA, 0.5µL Primer Foward A (10pM), 0.5µL Primer Reverse A (10pM), 0.5µL Primer Foward B (10pM), 0.5µL Primer Reverse B (10pM), 0.25µL Probe A (10pM), 0.25µL probe B (10pM).
?. Detection of FLU-A, FLU-B, PIV-1, PIV-3. For final volume reaction of 15µl, add 3µL cDNA, 0.5µL Primer Foward (10pM), 0.5µL Primer Reverse (10pM), 0.25µL Probe (10pM)
?. Detection of hBoV, AdV, β -Actin and RNAseP. For final volume reaction of 15µl, add 3µL cDNA, 0.25µL Primer Foward (10pM), 0.25µL Primer Reverse (10pM), 0.125µL Probe (10pM)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Adenosine-to-inosine (A-to-I) deamination is a functionally important modification of RNA that occurs in many metazoan nuclear transcripts, in several types tRNAs, and in mitochondrial transcripts of diplonemid protists. Several conceptually different methods exists to identify inosine residues in RNA, including the approach that exploits the cleavage after a guanosine or inosine residue by RNase T1. The digestion at Gs is blocked after an incubation in a glyoxal/borate solution and leads predominantly to cleavage 3′ to Is. This protocol blends the procedure originally conceived by Morse and Bass (DOI: 10.1021/bi9709607) and its more recent adaptation of Cattenoz <em>et al</em>. (DOI: 10.1261/rna.036202.112).

[BEFORE_START]
<ul>
<li>Prepare deionized glyoxal (see the protocol: <a href="https://www.protocols.io/view/Preparation-of-deionized-glyoxal-ejsbcne" target="_blank">Preparation of deionized glyoxal</a>).</li>
<li>Prepare 100 mM sodium phosphate buffer, pH 7.0.</li>
<li>Prepare 1 M sodium borate buffer, pH 7.5.</li>
<li>Prepare Tris-borate buffer (10 mM Tris-HCl, pH 7.8; 1 M sodium borate, pH 7.5).</li>
</ul>

[GUIDELINES]
<ul>
<li>The primer extension assay to map inosine residues achieves single-nucleotide resolution, but requires more material. The RT-PCR assay is more sensitive, but for single-nucleotide resolution, it needs to be coupled to 5′ adapter-ligation and sequencing. For a practical primer extension with sufficiently intense bands for cleavage intermediate detection, the target RNA in a sample should amount to &gt;0.1 pmol (&gt;10 ng of a 300 nt long transcript), optimally &gt;0.5 pmol.</li>
<li>The described procedure and quantities work well for a transcript, which represents 1-2% of the RNA sample. For the primer extension assay, if more RNA sample is required to achieve &gt;0.1 pmol of the target, scale up all components, so that guanosine residues in the sample RNA are evenly labeled with glyoxal-borate aducts.</li>
<li>If a purified or synthetic RNA is being analyzed, complement the sample to the final amount of 2.5 µg RNA with non-target RNA (e.g. total yeast tRNAs) to avoid over-digestion with RNase T1. Alternatively, for an RT-PCR assay, use 1 µg of poly(A) RNA as a substrate.</li>
</ul>

[STEPS]
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TITLE: Expression and purification protocol of GST-OPTN or (S177D, S473D)
AUTHORS: Chunmei  Chang, Chunmei Chang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the expression and purification protocol of GST-OPTN or (S177D, S473D).</div></div>

[STEPS]
?. [Protein expression]
Transform the E.Coli BL21DE3 cells with plasmid encoding for GST-OPTN and plated them on Amp plate.
?. [Protein expression]
Carry out the protein expression in  LB medium, induce with  IPTG (isopropyl- -d-thiogalactopyranoside) to an OD600 of 0.8 and grow at .
1.5 L
18 °C
?. [Protein expression]
Harvest the cells by spinning at  for  at  and stock at  until purification.
Centrifuge: 4500 34
4 °C
-80 °C
?. [Protein purification]
Follow the GST batch purification by Size Exclusion Chromatography.
?. [Protein purification]
Resuspend the pellets in Lysis Buffer, sonicate for cell lysis and clear at  at  for .
Centrifuge: 16000 33
4 °C
?. [Protein purification]
Incubate the supernatant with Glutathione Sepharose 4B (GE Healthcare) at  with gentle shaking for , apply to a gravity column, and wash extensively with Wash Buffer.
4 °C
?. [Protein purification]
Elute the protein of interest with Elution Buffer and then apply onto a Superdex 6 column (10/300 Increase) pre-equilibrated in SEC Buffer at .
4 °C
?. [Protein purification]
Pool the peak fractions containing pure protein, snap-frozen in liquid nitrogen and store at .
80 °C
TITLE: Minimal Basal Medium (liquid)
AUTHORS: Christa  Smith

[DESCRIPTION]
A defined artificial seawater medium for growing bacteria.

[BEFORE_START]
Acid wash all glassware with 10% HCl and rinse well with dH2O.

[GUIDELINES]
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.

[STEPS]
SECTION: Sea Salt Solution
1. Add 20 g Sigma Sea Salts into a 1 L glass bottle.
SECTION: Sea Salt Solution
2. Add 699 mL dH2O to sea salts in bottle and mix well to dissolve.
 
 
SECTION: Sea Salt Solution
3. Set prepared sea salt solution aside until ready to autoclave.
SECTION: FeEDTA Stock
4. Add 50 mg FeEDTA into a 250 mL glass bottle.
SECTION: FeEDTA Stock
5. Add 100 mL dH2O to FeEDTA in bottle and mix well to dissolve (stock concentration is 1.36 mM).
SECTION: FeEDTA Stock
6. Filter-sterilize FeEDTA solution using a 0.2 micron PES membrane filter.
SECTION: Basal Medium
7. Add 150 mL of 1 M Tris HCl, pH 7.5, into a 1 L glass bottle.
 
SECTION: Basal Medium
8. Add 0.34 g NH4Cl into the same bottle and dissolve.
SECTION: Basal Medium
9. Add 182.98 mg K2HPO4 into the same bottle and dissolve.
SECTION: Basal Medium
10. Add 375 mL dH2O to the same bottle and mix well.
11. Set prepared basal medium aside until ready to autoclave.
SECTION: Autoclave
12. Autoclave sea salt solution and basal medium.
 
 
 
SECTION: Autoclave
13. Cool autoclaved solutions to room temperature.
SECTION: Mix
14. Add 50 mL of FeEDTA stock to the bottle of sea salt solution and mix well.
SECTION: Mix
15. Add 250 mL basal medium to the bottle of sea salt solution and mix well.
SECTION: Mix
16. Add 1 mL of vitamin supplement the the bottle of sea salt solution and mix well.
 
SECTION: Finish
17. Store prepared medium at 4 Celsius.
 
SECTION: Carbon source
18. A carbon source can be added to the finished minimal medium.
TITLE: Detection of Batrachochytrium dendrobatidis (Bd) and amphibians DNA metabarcoding
AUTHORS: Omneya Ahmed Osman, Alexander Eiler, Mats Töpel, Tomas Larsson, Johan Andersson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">This protocol explain the qPCR assay for detecting </span><span>the pathogenic fungus Batrachochytrium dendrobatidis in environmental DNA samples and metabarcoding of amphibians species.</span></div></div>

[STEPS]
TITLE: JAX-Sen: Collection and shipment of specimen for single-cell RNA sequencing (scRNA-Seq)
AUTHORS: Laura Robinson, Susan Sheehan, Gaven Garland, Ron Korstanje

[DESCRIPTION]
These samples are part of the JAX-Sen project in the SenNet Consortium. Here we provide details on specimen collection and shipment to the Robson laboratory at The Jackson Laboratory for Genomic Medicine (JAX-GM) in Farmington, CT for its processing for single-cell RNA-sequencing (scRNA-seq).

[STEPS]
SECTION: Reagents and Materials:
1. 2mL Eppendorf tubes or 5 ml Eppendorf tubes
MACS tissue storage Solution (#130-100-008, MACS Miltenyi Biotech), 
cold storage (4 degrees Celsius)
Tweezers (clean, sterile)
SECTION: Quality Key Points:
2. The tissue specimen should be kept at 4 degrees Celsius and RNAse-free from its excision until it is processed at JAX. 
It is crucial to not store the tissue specimen at RT to avoid any cell death, tissue and/or RNA degradation. 
SECTION: Procedure:
3. Timeline: The daily shipping deadline at Jax BH is 12:00 noon. So, harvest the tissues before and closer to noon.
SECTION: Procedure:
4. Collection/Harvest:
SECTION: Procedure:
4.1. Prepare sterile tubes with cold MACS tissue storage solution (the volume will depend on the size of the specimen, but should cover abundantly the tissue and fill up the tube)
SECTION: Procedure:
4.2. Animal was euthanized via cervical dislocation. 
SECTION: Procedure:
4.3. Animal was pinned to a necropsy tray which remained on wet ice throughout the harvest. 
SECTION: Procedure:
4.4. Animal was perfused with 20ml cold PBS.
SECTION: Procedure:
4.5. Heart, pancreas, and kidney was collected and the specimen was transferred immediately into cold MACS tissue storage solution in tubes, on ice.
SECTION: Procedure:
4.6. Individually parafilm the Eppendorf tube lids to their tubes before shipping.
SECTION: Procedure:
4.7. Keep at 4 degrees Celsius thereafter, until shipping.
SECTION: Procedure:
5. Shipping:
SECTION: Procedure:
5.1. Place sample tubes in a plastic box (cardboard boxes insulate the samples from the cold ice) or in double Ziplock bags after checking that they are completely sealed.
SECTION: Procedure:
5.2. Ship the sample box on wet ice.
SECTION: Procedure:
5.3. Ship O/N on ice/ice packs, to:
The Jackson Laboratory for Genomic Medicine,
Farmington, CT, 06032
TITLE: microC-protocol
AUTHORS: Dimitrios Voukantsis, Kenneth Kahn, Martin Hadley, Rowan Wilson, Francesca M Buffa

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">microC is a multiscale virtual microenvironment for perturbation biology. It enables experiments that link genotype to phenotype taking into account the surrounding microenvironment. microC has a modular architecture that enables a wide variety of experiment. Furthermore, it offers easy access to advanced computational modelling and supercomputing resources to the wider scientific community.</div><div class = "text-block">microC combines agent-based and gene network modelling and uses partial differential equations to simulate interactions among cells. </div><div class = "text-block">The tool itself does not require any installation on the user’s machine, as it can be accessed using a web browser. Experimental results and data are also available via a web interface. This protocol describes the process of preparing and submiting an experiment with microC, and interpreting the simulation results</div></div>

[STEPS]
?. [Accessing microC]
microC is available via microc.org. During the first visit this step might take several seconds. This will not be the case in future visits.
?. [Preparing an experiment]
Experiments are specified via the microC main web page (Figure 1). The user may set a number of numerical parameters via sliders, specify the gene network, mutations, and parameters that control diffusion (please refer to the documentation page of microC for details, http://microc.org). microC modules are enabled through the specification files. For example setting parameters for Oxygen or any output node within diffusion-parameters.txt, will automatically enable a diffusion module for this substance. The "Run expreriment" buttom submits the expreriment to the computing cluster.
?. [Submitting an experiment]
Upon submission of the experiment (“Run Experiment” button, Figure 1) the user receives a link (Figure 2) that provides information about the status of the submission and the log file, where potential errrors are shown. Once the experiment has finished the same link will contain the results (that gradually become available in batches 16 replicates). Optionally, the user will be notified by email.
?. [Evaluating the results]
The results page displays data across all replicates and permits the viewer to inspect any of the replicates in detail. An animation of all replicates is displayed in the initial page along with averaged and detailed data for each of the experimental replicate (Figure 3). A more detailed presentation of the results page is available in the documation page of microc (http://microc.org).
TITLE: Electrophysiological recording of electrically-evoked compound action potentials
AUTHORS: James Fallon, Sophie Payne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Recording electrically-evoked compound action potentials (ECAPs) is performed to confirm that the electrode array is appropriately placed along the nerve. Recording ECAPS also allows confirmation that electrical stimulation is above neural threshold i.e. that electrical stimulation activates neural activity. The procedure is performed under anesthesia (immediately post-surgery) or in awake animals, and the procedure should incorporate all local requirements for standards of animal experimentation.</div></div>

[STEPS]
?. [Electrophysiological testing]
This procedure is performed immediately following implantation surgery or in awake, unrestrained animals. The electrical impedance of the array are tested using a custom stimulation to assess any open or short circuiting of wires.
?. [Electrophysiological testing]
Measure the impedance of each electrode using biphasic current pulses passed between the electrode of interest and all other implanted electrodes. Measure the peak voltage at the end of the first phase (Vtotal) of the current pulse following delivery of a 25 ms per phase current pulse and current of 931 mA.
?. [Electrophysiological testing]
If all electrodes are functional, connect a pair of electrodes to the stimulator and a pair of electrodes to the electrophysiological recording equipment
?. [Electrophysiological testing]
Record ECAPs similar to that described in:
?. [Electrophysiological testing]
Generate electrically-evoked compound action potentials (ECAPs) using bipolar stimulation and recording electrode configuration.
?. [Electrophysiological testing]
Make two sets of evoked electrophysiological recordings (averaged from a total of 50 responses).
?. [Electrophysiological testing]
Make recordings at currents from 0 to 2 mA in 0.1 mA steps using a biphasic pulse (width = 200 ms, 50 ms interphase gap) presented at a rate of 10 pulses per second.
?. [Electrophysiological testing]
Sample recordings at a rate of 100 kHz and filter (high pass: 200 Hz; low pass: 2000 Hz; voltage gain 103)
?. [Electrophysiological testing]
The electrically-evoked neural response threshold was defined as the minimum stimulus intensity producing a response amplitude of at least 0.1 mV
TITLE: qPCR assay for Aquarickettsia spp.
AUTHORS: Ana  Palacio, Grace Klinges, Stephanie Rosales

[DESCRIPTION]
qPCR for the quantification of Aquarickettsia spp. ( Klinges et al., 2022) a putative parasite found in the coral A. cervicornis. This protocol has been altered by incorporating a recently published A. cervicornis control gene (Palacio-Castro et al., 2021)  targeted to detect differences across A. cervicornis genotypes because it is a single-copy gene in A. cervicornis.




[STEPS]
SECTION: Prepare for qPCR
1. Remove PCR reagents from freezer and allow reagents to thaw on ice or at room temperature.
Wipe down PCR hood with bleach and ethanol. 
Place consumables such as tubes, plates, plate sealers, and water in PCR hood and turn on UV light for 20 min
Once everything is thawed vortex PCR reagents, spin them down, and place them on ice.
Keep reagents cool or on ice during the duration of the protocol. 
SECTION: qPCR thermocyler program settings
2. 
 




SECTION: Prepare PCR master mix
3. 

 
Add DNA last to each well 
Once the PCR master-mix reagents are combined, mix gently and spin down to collect mixture and remove bubbles that may interfere with downstream amplifications.
Place samples in thermocycler.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p style="text-align: center;"><img style="display: block; margin-left: auto; margin-right: auto;" src="https://s3.amazonaws.com/pr-journal/hnnfjtn.png" width="100" height="90" data-src="https://s3.amazonaws.com/pr-journal/hnnfjtn.png" data-ofn="nehirarma-üst-Tr.png" />  </p>
<p style="text-align: center;">Nehir Biyoteknoloji Ltd. <a href="http://www.nehirbt.com.tr/en/" target="_blank">www.nehirbt.com</a></p>
<p style="text-align: center;"> </p>
<p style="text-align: center;">This protocol describes the extreme high height microfluidic channel fabrication. In this case the height of the channels is 500 µm and this required some modifications on the previous protocol.</p>

[BEFORE_START]
<p>This protocol is derived from</p>
<p>[Reference: Processing guidelines for permanent epoxy negative photoresist SU8 2025, SU8 2035, SU8 2050 and SU8 2075, MicroChem company<strong>]</strong></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: U Mass - Alanine Transferase
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">
This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum alanine transferase levels reflect liver function.</div></div>

[STEPS]
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select ALT test on display and run the analysis.
?. Collect and analyze the data.
?. Perform daily quality control assessment of instrumentation before analysis.