eamag/protocols-with-steps huggingface dataset:
TITLE: Calculating number of Co-Migrated DNA Fragments across Pulsed Field gel electrophoresis (PFGE) profiles: Image analysis algorithm
AUTHORS: Ibrahim El-khalil Adam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The author describes a new image analysis algorithm that enables identification of how many DNA fragments co-migrate during PFGE.  The method is named  factor of co-migration based on exponential correlation between single-fragment bands and their pixel densities "FCM-ECSB".</div></div>

[STEPS]
?. [IMAGE PROCESSING]
Launch the program
?. [IMAGE PROCESSING]
Open PFGE image of interest. You should navigate to containing folder first.
?. [IMAGE PROCESSING]
It is critical to tell the program if DNA bands are white in a black background or otherwise.
?. [IMAGE PROCESSING]
Define lanes and then bands.sometimes, manual modification is necessary.
?. [IMAGE PROCESSING]
Select the lane that shows the DNA ladder. then indicate each band with its corresponding size. (Sizes are in kilo base-pair (kbp). you should multiply each value by 1000 to get accurate results).
?. [IMAGE PROCESSING]
CALULATING EXPECTED PIXEL DENSITIES FOR CO-MIGRATED DNA FRAGMENTSThis step is critical. it is based on end-user's estimation. Keep in mind that co-migrated fragments show high pixel densities (Intensity profiles). band intensities should reduce as you go down the profile (toward smaller band sizes).
?. [IMAGE PROCESSING]
Copy all the data from "Analysis info." from GelAnalyzer2010a. Press Ctrl + A to copy data.
?. [IMAGE PROCESSING]
Open PAST statistics. The goal is to get a polynomial equation with significantly high R2 value.  Past the data copied from GelAnalyzer2010a and past it into PAST statistics. Change column attributes to indicate (Band size) and (Pixel density).
?. [IMAGE PROCESSING]
Create a preliminary polynomial fit. R2 value at this point roughly indicates number of co-migrated fragment across the profile.
?. [IMAGE PROCESSING]
Refer to step 2 (remove values above the red line). Recreate the polynomial fit and check R2 value. Remove band sizes and their corresponding pixel densities from both columns (According to ODD values of pixel densities). The polynomial equation we need is:EPD = -6.651E-11*BS^2+0.0013*BS +191.2EPD: expected pixel density, BS: band size
?. [IMAGE PROCESSING]
DENOTATION OF ALL BAND SIZES INTO TE EQUATION (from step 2.4):This step requires the same two columns from step 2.2. Open Microsoft Office™ Excel. Past the two previously mentioned columns. Start denotation of column "Band size" into the equation. Use an empty column.
?. [IMAGE PROCESSING]
In order to calculate approximate number of DNA fragments represented by each PFGE band, we must simply divide observed PDs by the corresponding ones. column is named "O/E" indicating observed/expected.
?. [IMAGE PROCESSING]
FCMs are simply truncated values of O/E column.Here is a screen-recorded video showing the entire protocol in action :
TITLE: Immunofluorescence Assay to detect respiratory syncytial virus
AUTHORS: Maria Lucia Silva, Talita Gagliardi, Eurico Arruda

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">IF Protocol used to detect RSV in nasopharyngeal samples.</div></div>

[STEPS]
?. Fix the cells immerge them into a pure cold acetone for 5min.
?. Wash 3X the sample in PBS for 3min each and at room temperature (r.t.).
?. Permeabilize the cells incubate with 0.1% Triton-X100 for 15min at r.t.
?. Wash 3X the sample in PBS for 3min each and at r.t.
?. Incubate the cells with 1:100 dilution of “anti-RSV blend monoclonal antibody” (MAB858-4; Millipore, MA, USA) in PBS with 1% Bovine Serum Albumin (BSA) and 0.2% Tween 20. Keep it inside a humidified chamber for 30min at r.t.
?. Wash 3X the sample in PBS for 5min each and at r.t.
?. Incubate the sample for 15min at r.t in n a Block solution (10% of specific serum diluted in PBS 1X).
?. Wash 3X the sample in PBS for 3min each and at r.t.
?. Incubate the cells with the secondary antibody solution for 30min at r.t. and inside a humidified chamber. It was used “Donkey anti-mouse IgG (H+L) secondary antibody conjugated with Alexa Fluor® 488” (Life Technologies, Thermo Scientific, Carlsbad, CA, USA) diluted 1:200 in PBS with 1% BSA.
?. Wash 3X the sample in PBS for 5min each and at r.t.
?. Incubate the cells with 5µg/ml of DAPI solution (diluted in PBS) for 5min at r.t. and inside a humidified chamber.
?. Wash 2X the sample in PBS for 5min each and at r.t.
?. Mount coverslip with a drop of mounting medium (90% glycerol diluted in PBS).
?. Seal coverslip with nail polish to prevent drying and movement under microscope.
?. Store in dark at -20°C or +4°C.
TITLE: Attention! Simpli Health ACV Keto Gummies Reviews: Is SimpliHealth ACV Keto Works- Cost, Side Effects, Ingredients, Benefits, Where To Buy?
AUTHORS: johnkikal  

[DESCRIPTION]
Attention! Simpli Health ACV Keto Gummies Reviews: Is SimpliHealth ACV Keto Works- Cost, Side Effects, Ingredients, Benefits, Where To Buy?

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A method to load algal cells with dextran-conjugated fluorescent dyes. This is a good way of testing and optimising conditions for biolistic DNA delivery.</p>

[STEPS]
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TITLE: FFPE RNA Quality Evaluation (Qiagen) -- University of Minnesota TMCs
AUTHORS: Qiagen, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Completed prior to going through -omics and spatial technologies to evaluate FFPE blocks for their RNA quality. 

[BEFORE_START]
Evaluate if this is required for processing with your platform/technology. Not all require a certain DV200 to move forward with processing. 

If tissue sample is scarce or rare, consider the best step -- Use some of the sample for RNA quality evaluation or move forward without it, understanding the risks and/or benefits of doing so. 

[STEPS]
2.   

4.   

SECTION: Preparation of Samples for Processing
3. Create or request curls from the FFPE blocks (typically done by histologist). 
Follow recommendations as noted below. 
Keep curls in a 1.5mL tube at -80ºC until RNA isolation process. 
1. Qiagen Protocol: 
  

Qiagen product website:  https://www.qiagen.com/us/products/discovery-and-translational-research/dna-rna-purification/rna-purification/total-rna/rneasy-ffpe-kit 
Cat. No. / ID: 73504

Qiagen Support contact form: https://www.qiagen.com/us/knowledge-and-support/~/link.aspx?_id=5402A118CD214AF7ADBAC65422659A71&_z=z

Time needed: ~2-4 hours, depending on number of samples 
SECTION: Once RNA isolation complete
5. Complete: 
Nanodrop for RNA quantification 
DV200 for (Eukaryotic total) RNA qualification
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a storage solution that stabilizes and protectsRNA while inactivating RNase

[GUIDELINES]
<strong>Note:</strong> If you read the patent application, they refer to an ammonium sulfate concentration of 700g per L. What they really mean is add 700g to 1L (including EDTA and sodium citrate at conc. listed above), so you end up with more than 1L total volume. <br /><br /><strong>Note:</strong> The pH should be ~7 using this recipe. The patent states that pH can range from 4-8; the preferred commercial pH is 5.2; I use pH 7 for bacterioplankton samples, but pH can be lowered by adding H<sub>2</sub>SO<sub>4</sub>.<br /><br /><strong>Note:</strong>  Since this is a storage solution for RNA, use high quality reagents ("molecular biology" grade) when preparing the buffer.

[STEPS]
?. 
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?.
TITLE: Removal of genomic DNA from RNA preparations (Thermo Scientific )- (M4455 Version)
AUTHORS: Maximilian Dietsch

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Removal of genomic DNA from RNA preparations</div></div>

[STEPS]
?. AB1Add to an RNase free tube:&nbsp;2RNA1 µg310X reaction&nbsp;buffer&nbsp;with&nbsp;MgCl21 µl4DNase I, RNase-free1 µl (1U)5Waterto 10 µl
AB1Add to an RNase free tube:&nbsp;2RNA1 µg310X reaction&nbsp;buffer&nbsp;with&nbsp;MgCl21 µl4DNase I, RNase-free1 µl (1U)5Waterto 10 µl
?. Incubate at 37 °C for 30 min
?. Add EDTA, Water and PCI and vortex thoroughly.
[EDTA]
[Water]
[PCI (phenol chloroform isoamyl alcohol)]
?. Centrifuge for 10 min at 10000 rpm and 4 °C
?. transfer the upper phase into a fresh tube and add 3 volumen EtOH/ 3M Natrumacetat  (30:1, ph 5.2 )
?. precipitate RNA over night at -20 °C
?. Centrifuge 30 min at 13000 rpm and 4 ° C
?. Discard supernatant and wash pellet with 75% EtOH ( do not resuspend the pellet)
?. Centrifuge 10 min at 13000 rpm and 4 °C
?. [repeat washing step]
[Centrifuge]
?. Discard supernatant and dry pellet for 10 - 15 min
?. resupend pellet with 30 µl H20
TITLE: Condor CBD Gummies Are 100% Real &amp; Effective and Available In A Variety Of Flavors.
AUTHORS: Condor CBD Gummies Review

[DESCRIPTION]
 
     Condor   CBD Gummies: Provide rapid relief from health issues and enhance mental   clarity. Effectively cures joint. It relaxes your nerve cells, which gives   you the relaxation great product to relieve stress and anxiety. 
 

[STEPS]
TITLE: Yeast two-hybrid interaction assay
AUTHORS: Mélanie Bonhivers, Anne-Lise Sahin, Nicolas LANDREIN

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein-protein interaction can be tested using yeast two-hybrid assays. The bait protein is fused to the Gal4 DNA-binding domain (BD) and the prey is fused to the  Gal4 transcriptional activation domain (AD) are both expressed in the same yeast cell. Activation of the reporter genes occurs only if bait and prey interact.



</div></div>

[STEPS]
TITLE: Creolimax fragrantissima_culture method
AUTHORS: Meritxell  Antó

[STEPS]
?. [Growing medium]
BD Difco Marine Broth 2216 • Marine Agar 2216 https://legacy.bd.com/europe/regulatory/Assets/IFU/Difco_BBL/212185.pdf
?. [Growth conditions]
Temperature 12ºC
?. [Cryopreservation and recovery (from liquid cultures)]
Cryopreservation  (10%DMSO in Growing medium)1. Pre-grow the culture in appropriate growing medium until almost saturated but not too old.2. Mix 1.35mL of the culture with 150 ul of DMSO  in the screw-top cryovials.3. Transfer the cryovials to the Freezing container, Nalgene®Mr. Frosty unit. Place it in the -80ºC freezer and leave it overnight.4. Remove cryovials from the Freezing container and store in appropriate boxes for -80ºC.Recovery1. Fill a flask with 10mL fresh Growing medium.2. Take out the frozen stock from the freezer, carry it to the culture hood in a cryo box to prevent it from thawing.3. With a pipette tip (or a loop), take a chunk of the frozen stock and resuspend it in the culture flask (~10uL is probably sufficient). Take the frozen stock back to the -80ºC as rapidly as possible.4. Let it recover for at least 3-4 days and proceed with another transfer in a fresh medium even if the cells are not completely grown it will help to dilute the remaining DMSO from the cryopreservation, the time will vary depending on the amount of the initial inoculum and the culture type.*NOTE: because of DMSO some precipitates may appear in the culture.All protists must be stored in liquid nitrogen for long-term storage. Although storage at -80°C is sometimes sufficient for a temporary alternative.
TITLE: HPF-FS of Solarion arianae for transmission electron microscopy
AUTHORS: Marek Valt

[DESCRIPTION]
This is an optimized version of the protocol for standard High Pressure Freezing followed by Freeze Substitution (HPF-FS) for transmission electron microscopy that was used for the fixation of the culture of Solarion arianae.

[STEPS]
SECTION: Cell harvest
1. Centrifuge a well-grown culture at 1500 g at 4 °C.
SECTION: Cryo-immobilization by high-pressure freezing
3. After high-pressure freezing, the samples were transferred to precooled (-90 °C) fixative medium (2% OsO4 in 100% acetone) and processed by automatic freeze substitution (Leica EM AFS), as follows:
SECTION: Cell harvest
1.1. Discard supernatant and collect pellet.
SECTION: Cryo-immobilization by high-pressure freezing
3.1. incubation at -90 °C for 96 hours
SECTION: Section preparation
8. Cut sections. 
For Solarion HPF-FS, 90nm thick sections were cut with a diamond knife on an an EM UC6 (Leica) ultramicrotome.
SECTION: Section preparation
9. post-contrast with uranyl acetate and lead citrate.
SECTION: Cryo-immobilization by high-pressure freezing
2. The obtained pellet was cryo-immobilized by high-pressure freezing (Leica EM ICE) in gold-plated, 3 mm wide copper carriers with a cavity of 0.1 mm (Wohlwend GmbH).
SECTION: Cryo-immobilization by high-pressure freezing
3.2. warm up to -20 °C (5°C/hour for 14 hours)
SECTION: Cryo-immobilization by high-pressure freezing
3.3. incubation at -20 °C for 24 hours
SECTION: Cryo-immobilization by high-pressure freezing
3.4. warm up to 4°C (3°C/hour for 8 hours)
SECTION: Cryo-immobilization by high-pressure freezing
3.5. Incubation at 4°C for 18 hours
SECTION: Cryo-immobilization by high-pressure freezing
4. After AFS, the samples were transferred to room temperature and washed with 100% acetone (3x).
SECTION: Cryo-immobilization by high-pressure freezing
5. After washing, infiltrate sequentially for 1h each with: 
SECTION: Cryo-immobilization by high-pressure freezing
5.1. resin-acetone mixture 1:2
SECTION: Cryo-immobilization by high-pressure freezing
5.2. resin-acetone mixture 1:1
SECTION: Cryo-immobilization by high-pressure freezing
5.3. resin-acetone mixture 2:1
SECTION: Cryo-immobilization by high-pressure freezing
6. Finally, the samples were infiltrated with pure resin overnight (EMbed 812)
SECTION: Cryo-immobilization by high-pressure freezing
7. Lastly, polymerize for 48 hours at 60 °C
SECTION: Section preparation
10. Lastly, coat with a carbon layer.
TITLE: An overview of growth factors as the potential link between psoriasis and metabolic syndrome. 
AUTHORS: Mateusz Matwiejuk, Hanna Myśliwiec, Adrian Chabowski, Iwona Flisiak

[DESCRIPTION]


Psoriasis
is a chronic, complex, immunologically mediated, systematic disease that can
not only affect the skin but also the joints, and nails. It may coexist with
various other disorders such as depression, psoriatic arthritis, cardiovascular
diseases, diabetes mellitus, and metabolic syndrome. In particular, the
potential link between psoriasis and metabolic syndrome is worthy of an
attention issue. A dysregulated level of growth factors could potentially
contribute to the disturbances of keratinocyte proliferation, inflammation, and
severity of itch. However, the
pathophysiology of psoriasis and its comorbidities, such as metabolic syndrome,
remain incompletely elucidated. Growth factors and their abnormal metabolism may
be the potential link connecting this conditions. Overall, the objective of
this review is to analyze the role of growth factors disturbances in psoriasis
and in metabolic syndrome.




[STEPS]
9. Records
identified 

1. Identification of records. Databases (n = 1) Pubmed
2. Screening of records

Records screened (n = 1579)


3. Records excluded (n = 1372 )
No data available
Abstracts
4. Reports assesed for eligibility (n = 207 )
5. Reports excluded 

Reason 1 (n = 38 ) Duplications

Reason 2 (n = 77 ) Out of scope 

6. Studies included in review  (n = 92 )
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><u>Marchantia protoplast preparation (adapted from </u><u>Bopp and Vicktor 1988)</u></p>

[BEFORE_START]
<p>Perform surface sterilization of Marchantia spores (see Step 1-7 in protocol “Agrobacterium mediated transformation of Marchantia spores” by Dr. Susana Sauret-Gueto). Let sporelings grow for 10-14 days post surface sterilization.</p>

[STEPS]
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TITLE: Plasmid Construction and Gibson cloning
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes plasmid construction.

[STEPS]
SECTION: Plasmid Construction
1. Obtain the sequences of all cDNAs by amplifying existing plasmids, HAP1 cDNA, or through gene synthesis (Genscript).
SECTION: Plasmid Construction
2. For insect cell expressions, the sequences are codon optimized and the gene is synthesized (Genscript).
SECTION: Plasmid Construction
3. With the exception of the NAP1-6xAla mutant, which was obtained through gene synthesis (Genscript), all other plasmids were generated by Gibson cloning. 
SECTION: Plasmid Construction
4. For Gibson cloning, generate inserts and vector backbones by PCR amplification or excise from agarose gels after restriction enzyme digestion at 37 °C for 120 min.
SECTION: Plasmid Construction
5. Purify the inserts and plasmid backbones with Promega Wizard SV gel and PCR Cleanup System (Promega).
SECTION: Plasmid Construction
6. Mix the purified inserts and backbones in a molar 3:1 ratio, respectively, supplemented by a 2x NEBuilder HiFi DNA assembly enzyme mix (New England Biolabs).
SECTION: Plasmid Construction
7. Incubate Gibson reactions for 60 min at 50 °C and then transform into DH5-alpha competent E. coli cells.
SECTION: Plasmid Construction
8. Grow transformed Gibson reactions 60 min on agar plates containing the appropriate selection marker (ampicillin, kanamycin, or chloramphenicol).
SECTION: Plasmid Construction
9. Pick single colonies, grown 60 min in liquid cultures, and pellet for DNA plasmid extraction using the GeneJet Plasmid Miniprep kit (Thermo Fisher).
SECTION: Plasmid Construction
10. Submit the purified plasmid DNA for DNA Sanger sequencing (MicroSynth AG) to verify insert sequences by Sanger sequencing. 
SECTION: Plasmid Construction
11. Further analyze positive clones by whole plasmid sequencing (Plasmidsaurus).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Fluorescent activated cell sorting (FACS) is a specialized type of flow cytometry used for sorting and analyzing a heterogeneous mixture of cells into different subpopulations based on the specific light scattering and fluorescent characteristics (from the specific labels) of each cell. The number of measurable parameters that can be used by this technology to separate cell populations is immense – starting from simple surface immunophenotyping to metabolic functions, cell cycle status, redox state, and DNA content analysis to name a few.</p>
<p><br />Since its inception, FACS has been used extensively in biomedical research and clinical diagnostics and therapeutics. The most common usage of FACS is seen in:<br />- Analysis of whole human blood for diagnosing diseases, immunophenotyping<br />- Sorting different blood cell fractions for ex-vivo manipulations and/or transplantations<br />- Immuno-phenotypic analysis of murine blood to identify transgenic/knockout animals<br />- Sorting and analysis of a slew of cell lines for various biological assays<br />- Characterization and isolation of rare cells types like adult stem cells and cancer initiating cells </p>
<p> </p>
<p>Each human cell expresses hundreds of thousands of cell surface antigens that specify their cell type, biological function, development stage, and much more. Cells residing in different organs have characteristic cell surface antigens, and determination of these cells using the specific fluorophore-conjugated antibodies can be analyzed by flow cytometry. The following general protocols are recommended for various common FACS staining procedures. Staining with unconjugated purified antibody needs an additional step of staining with a fluorescent conjugated secondary antibody (indirect immunostaining).</p>

[GUIDELINES]
<p>This information is to serve as a guide as individual investigators may need to optimize protocols for their particular cell type.</p>

[STEPS]
?. 
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TITLE: Nephele DADA2 Workflow
AUTHORS: Nandie Elhadidy

[DESCRIPTION]
A protocol outlining the workflow of utilizing DADA2 on Nephele with a provided practice dataset.  DADA2 is a pipeline that provides species assignments using Amplicon Sequence Variants or ASVs which compare variants to a predetermined sequence through assumption that the most amplified sequence is the correct one. 


[STEPS]
SECTION: Downloading the Dataset
1. To download the practice dataset provided by Nephele, visit: https://nephele.niaid.nih.gov/user_guide_data/
SECTION: Running DADA2
4. Click the Home tab.
2. Download the first set of sequences listed as well as the mapping file. 
SECTION: Running DADA2
5. Choose the Analyze option.
SECTION: Analysis of the Output
13. From the My Workspace tab, hover to the view results icon under Actions for the completed DADA2 job. 
SECTION: Analysis of the Output
14. Under Reported Statistics, Reported Files, Reported Images, Reported Tables, and Reported Charts and explore outputs. 
SECTION: Running DADA2
6. Under Amplicon Metagenomics, select "Start job" option under DADA2.
3. Once downloaded, unzip the downloaded file and ensure all .fastq.gz files are present. 
2.1. The reference provided for this set of sequences is listed as: Experimental Microbial Dysbiosis Does Not Promote Disease Progression in SIV-Infected Macaques. NCBI BioProject: PRJNA417022
SECTION: Running DADA2
7. Select Paired End FASTQ, as this is a paired-end dataset.
SECTION: Running DADA2
8. Click "Upload from my computer" and add all .fastq.gz files from the downloaded dataset. Start upload
SECTION: Running DADA2
9. Select "Next" and upload the corresponding Mapping File. 
SECTION: Running DADA2
10. After reaching the Pipeline Selection page, click Select and enter a description of the job.
SECTION: Running DADA2
11. Keep Filter and Trim, Merge Pairs, and Analysis settings defaulted. Do not check Ion Torrent Data. Click Validate and Submit *Note: This is for following the exact tutorial instructions. If you would like to make changes to these pre-sets, proceed with these changes. 
SECTION: Running DADA2
12. Nephele will send an email upon job completion.
SECTION: Analysis of the Output
14.1. Reported files of note include: Rooted tree, Rarefaction curve, Alpha diversity, PCoA binomial, BCoA Bray-Curtis, and ASV heatmap. 
 
SECTION: Analysis of the Output
14.3. Reported tables of note include: Read counts by sample and Top species table
SECTION: Analysis of the Output
14.2. Reported images of note include: Quality profiles of forward and reverse reads
SECTION: Analysis of the Output
14.4. Reported charts of note include: Reads in vs. reads out and Track reads
TITLE: Labelled Amino Acids
AUTHORS: BOC      Sciences

[DESCRIPTION]
Isotope-labeled amino acids refer to the replacement of single or multiple atoms of the amino acid with isotopes, which can be radioactive isotopes or stable isotopes. Basically, isotope-labeled amino acids have identical structures and properties as non-labeled amino acids. The trends of isotopes-labeled amino acids can be observed by analytical methods such as mass spectrometry and nuclear magnetic resonance. Therefore, isotope-labeled amino acids serve as safe, effective, and convenient tracer tools for scientific research in medicine, biology, pharmacy, chemistry,etc.

In the field of medicine: Isotope-labeled amino acids can be used for the clinical diagnosis of various diseases. Labeled amino acids can be used as tracers to track the metabolic processes of amino acids and proteins in different physiological and pathological conditions, which allows the exploration of disease mechanism and effective ways to prevent and treat diseases.
In the field of biology: Isotope-labeled amino acids are used as starting materials to study the biosynthetic pathways and mechanisms of natural products in animals, plants, and microorganisms by analyzing the abundance of labeled elements in products.
In the field of pharmacy: Isotope-labeled amino acids are critical tools for the development of new drugs, and are used to determine the transfer, transformation, efficacy, mechanism of action, toxicity, and side effects of drugsin vivo.
In the field of chemistry: Isotope-labeled amino acids can be used as tracers to explore chemical reaction mechanisms and as raw materials for the synthesis of other labeled products, such as labeled polypeptides.

[STEPS]