eamag/protocols-with-steps huggingface dataset:
TITLE: Cell Surface Flow Cytometry Staining of Whole Blood
AUTHORS: Sam Li

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

[STEPS]
?. Add predetermined optimum concentrations of desired fluorochrome conjugated, biotinylated, or purified primary antibodies to 100µl of anti-coagulated whole blood.
?. Incubate at room temperature for 15-20 minutes in the dark.
?. Dilute 10X Red Blood Cell (RBC) Lysis Buffer (BioLegend Cat. No. 420301) to 1X working concentration with DI water. Warm to room temperature prior to use. Add 2ml of 1X RBC lysis solution to whole blood/antibody mixture. Incubate at room temperature for 10 minutes.
?. Centrifuge at 350xg for 5 minutes, discard the supernatant.
?. Wash 1X with at least 2ml of Cell Staining Buffer by centrifugation at 350xg for 5 minutes.
?. If using a purified primary antibody, resuspend pellet in residual buffer and add a previously determined optimum concentration of anti-species immunoglobulin fluorochrome conjugated secondary antibody (e.g. FITC anti-mouse Ig) and incubate in the dark for 15-20 minutes.
?. If using a biotinylated primary antibody, resuspend cell pellet in residual buffer and add a previously determined optimum concentration of fluorochrome conjugated Streptavidin (SAv) reagent (e.g. SAv-PE, BioLegend Cat. No. 405204) and incubate for 15-20 minutes in the dark.
?. Repeat step 5.
?. Resuspend cells in 0.5ml Cell Staining Buffer or 0.5ml 2% paraformaldehyde-PBS fixation buffer.Tip: For gentler fixation (particularly with tandem fluors), FluoroFix™ Buffer (Cat. No. 422101) may be used.
?. Perform fluorescence activated cell sorting (FACS), or flow cytometric analysis.Note: If you are unable to immediately read your samples on a cytometer, keep them shielded from light and in a refrigerator set at 4-8°C. The samples should be resuspended in Cell Staining Buffer. Note that samples should not remain in a fixation buffer for extended periods of time as this can affect fluor conformation and fluorescence.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Se realiza la extracción de la fracción rica en leucocitos a partir de sangre total usando la sedimentación de los eritrocitos en una solución con carboximetil celulosa e iodioxanol al 12%. La extracción de los PMNs a partir de dicha fracción en un gradiente discontinuo de densidad de 1,077- 1,095 g/ml. Basado en el protocolo de la empresa Axis-shield application sheet C11 usando optiprep (iodioxanol 60%). (isolation of human plymorphonuclear leukocytes (granulocytes) from a leukocyte rich plasma in a discontinuous iodioxanol gradient).</p>
<p> </p>
<p>La realizacion de este protocolo fue posible gracias al apoyo del departamento administrativo de ciencia tecnología e innovacion, Colciencias a traves del proyecto 111356933173 convocatoria569-2012.</p>

[BEFORE_START]
<p>La preparación de los reactivos se debe hacer por lo menos con un día de anterioridad, principalmente porque la carboximetil celulosa demora mucho tiempo para diluirse. </p>

[GUIDELINES]
<p>La purificacion de PMNs se basa en la extraccion de la fracción leucoplaquetaria y posterior extracción de los PMNs en un gradiente discontinuo de densidades de 1.095gr/mL y 1.077gr/mL. </p>

[STEPS]
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?.
TITLE: qPCR standard for library quantification
AUTHORS: Ayinuer Aximu Petri, Matthias Meyer

[DESCRIPTION]
Protocol for the preparation of a standard for DNA library quantification by quantitative real-time PCR (Meyer et al. 2008; Gansauge et al. 2020). 

References

Meyer, M., Briggs, A. W., Maricic, T., Höber, B., Höffner, B., Krause, J., Weihmann, A., Pääbo, S., & Hofreiter, M. (2008). From micrograms to picograms: Quantitative PCR reduces the material demands of high-throughput sequencing. Nucleic Acids Research, 36(1): e5.

Gansauge, M.-T., Aximu-Petri, A., Nagel, S. et al. Manual and automated preparation of single-stranded DNA libraries for the 
sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols,15, 2279-2300 (2020).


[STEPS]
TITLE: Protein Concentration Determination using Qubit
AUTHORS: Steven Burgess

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Procedure for quantification of protein concentration. This version is adapted for use with samples that have been extracted in protein extraction buffer as part of processing soybean/cowpea samples, but the general outline is similar for other samples. See the manual for further details:  </span><a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Qubit_Protein_Assay_UG.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Qubit_Protein_Assay_UG.pdf</span></a></div></div>

[STEPS]
?. [Create Working Solution for Analysis]
Create a working solution of Qubit assay buffer by diluting the reagent 1:200 in the provided buffer.
The final volume in each tube must be 200 µL. Each standard tube requires 190 µL of Qubit® working solution, and each sample tube requires anywhere from 180–199 µL. Prepare sufficient Qubit® working solution to accommodate all standards and samples.
?. [Create Sample Dilution for Analysis]
Dilute sample 1:50 ( dH2O +  sample)
196 µl
4 µl
After taking into consideration the Qubit dilution factor (1:20; 10 μL sample + 190 μL qubit assay buffer) the sample being analyzed has been diluted 1:1000. This should give a value in the linear range for qubit (1.25-25 μg/mL). This should also reduce the impact of SDS on the quantification, which must be >0.2% in the 10 μL sample added (conc SDS in PEB is 2 %, so diluted 1:50 yields 0.04 %)
?. [Create Sample Dilution for Analysis]
Add  of Qubit working solution to a fresh Qubit assay tube, one for each sample to be analyzed (including the three protein standards)
190 µl
?. [Create Sample Dilution for Analysis]
Add  of protein standard to the appropriate tube and mix by vortexing s.
10 µl
?. [Create Sample Dilution for Analysis]
Add  of diluted sample to the appropriate tube and mix by vortexing  s.
10 µl
?. [Create Sample Dilution for Analysis]
Allow samples to incubate at room temperature for
?. [Create Sample Dilution for Analysis]
Measure protein sample concentration using the Qubit, following the instructions on the machine (i.e. start by measuring the standard curve).
Remember to adjust measured values according to the dilution factor applied. In this example, after taking into consideration the Qubit dilution factor (1:20;   sample +    Qubit assay buffer) the sample has been diluted 1:1000
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol outlines how to generate tables to analyze CRISPR interactions. Based on methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Supplemental information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<span class="identifier">sessionInfo</span><span class="paren">(</span><span class="paren">)<br /><br />## R version 3.2.0 (2015-04-16) <br />## Platform: x86_64-apple-darwin13.4.0 (64-bit) <br />## Running under: OS X 10.10.4 (Yosemite) <br />## ## locale: <br />## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8 <br />## <br />## attached base packages: <br />## [1] stats graphics grDevices utils datasets methods base <br />## <br />## loaded via a namespace (and not attached): <br />## [1] magrittr_1.5    formatR_1.2    tools_3.2.0    htmltools_0.2.6 <br />## [5] yaml_2.1.13    stringi_0.4-1    rmarkdown_0.7    knitr_1.10.5 <br />## [9] stringr_1.0.0    digest_0.6.8    evaluate_0.7</span>

[STEPS]
?. 
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?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a DNA extraction protocol adapted from Byron Crump's lab, which was developed from both Zhou et al. 1996 and Crump et al. 2003. It is intended to be used with 47 mm 0.22 um PES filters or a 0.22 uM Sterivex filter, both preserved with RNALater after sample collection.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
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?. 
?.
TITLE: Donor eligibility Criteria for Human Liver for HuBMAP TTD-Columbia/PSU
AUTHORS: Presha  Rajbhandari, Hua   Tian, Helen   Remotti, Brent    Stockwell, bs  

[DESCRIPTION]
This documents outline the criteria for human liver tissue donor eligibility for the Hubmap BioMolecular Atlas Program (HuBMAP), Transformative Technology Development (TTD) Center at Columbia University/Pennsylvania State University. Samples include those from liver resections/biopsies/transplantation. Donor eligibility, consent for tissue donation, documentation on donor medical history and sample storage is performed by the Columbia University Biobank.

[STEPS]
SECTION: Inclusion criteria for Donor:
1. 
-Age range >18
-BMI <29
-All race, ethnicity and gender (equal male and female) are accepted
-Negative for HIV, Hepatitis B or C virus, CMV or EBV, COVID-19, sepsis
-Negative for liver diseases including fatty liver disease, hepatocellular carcinoma, and diabetes
SECTION: Sample:
2. -Visual inspection of liver for any abnormalities
-Site of tissue far from benign lesions for resection cases
-If available, recover at least 1cm x 1cm x 1cm of liver and flash freeze as quickly as possible on dry ice /isopentane slurry (without OCT) and store at -80C
-Warm ischemia time <30 minutes and/or cold ischemia time < 2 hours
-Normal histology and tissue quality assessed by hematoxylin and eosin staining by expert pathologist
SECTION: Sample information:
3. The metadata related to the sample to include:
-Pathogenesis/diagnosis for resected tissue or donor cause of death
-Donor age
-Demographics
-Medications and dose
-Serological test results including HepB, HepC, HIV, CMV, EBV
-Past medical history
-Smoking/Drug use/Alcohol use
-Time from brain death, if deceased liver donors
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<div>
<div>
<div>
<p>This assay was designed and developed by Alyssa Pyke and Bruce Harrower at this laboratory aided by design software in use at the time.</p>
<p>The assay specifically targets the haemagglutinin (HA) region of influenza A(H1)pdm09 virus strains and is designed as a qualitative screening test for human cases of infection, but not for infection due to other known influenza viruses.</p>
<p>The assay aims to detect any circulating seasonal H1N1 influenza strains, not only influenza A(H1)pdm09 virus.</p>
</div>
</div>
</div>
</div>
</div>

[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<div>
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol and with PCR in general.</li>
</ul>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
?. 
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?.
TITLE: Official website:-https://wintersupplement.com/instahard-reviews/
AUTHORS: Exipure  Reviews

[DESCRIPTION]
InstaHard Reviews

[STEPS]
1. Official website:-https://wintersupplement.com/instahard-reviews/
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TITLE: ALS and the Gut-Brain Axis: A protocol for a systematic review and meta-analysis assessing the relationship between amyotrophic lateral sclerosis, the gut and its microbiome.
AUTHORS: Alexander T Jackson, Rollo G. Press, fergal waldron, Jenna Gregory

[DESCRIPTION]
Abstract

Background: ALS is a devastating neurodegenerative disease characterized by progressive loss of motor function. At the time of symptom manifestation, effective treatment options are extremely limited, due in part to the irreversible loss of motor neurons at this late stage of the disease. Recent research has highlighted the involvement of both gut pathology and gut microbiota as important factors in early disease pathogenesis, which may represent an opportunity for early diagnosis and intervention, prior to neurological symptom onset and thus prior to significant motor neuron loss. 

Aim: The aim of this review is to perform a comprehensive analysis of the literature to identify potential pre-symptomatic pathological hallmarks of ALS in the gut, including, but not limited to, changes in gut microbiota and protein aggregation. 

Methods: This protocol details the strategy for the proposed systematic review and meta-analysis to identify and compare studies assessing gut-related pathological changes in ALS separated in to two analyses assessing (i) gut microbiota and (ii) non-microbiota related pathways, with the ultimate aim of identifying opportunities for early disease detection before neurological symptom onset.

[STEPS]
TITLE: Summary of the housing and husbandry conditions of Zebrafish (Danio rerio) at University of Glasgow
AUTHORS: Anita Racz, Brooke Allan, Toni Dwyer, Davide Thambithurai, Amélie Crespel, Shaun S. Killen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The environmental and housing conditions which fish experience may have profound effects on their biology, physiology and behaviour. As so, it is essential to share the housing and husbandry details of laboratories in scientific papers. Unfortunately, most of the times, these details are overlooked and just referred as „standard holding conditions”. As these conditions can have strong effect on the animals and could be different between and within facilities (even between different rooms in the same facility), the absence of housing and husbandry details in scientific papers could limit the reproducibility of the studies. To be as informative as possible, we are thus providing the details of the housing and husbandry conditions of zebrafish (</span><span style = "font-style:italic;">Danio rerio</span><span>) used in projects at the University of Glasgow during 2017 – 2020. Details about our earlier zebrafish housing conditions can be found in our previous paper (Rácz et al., 2019- </span><a href="https://doi.org/10.1089/zeb.2018.1628" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">doi.org/10.1089/zeb.2018.1628</span></a><span>).</span></div></div>

[STEPS]
?. [Housing conditions]
The detailed information on the housing and husbandry conditions can be find in the attached PDF:
TITLE: Procedures for Performing the Mixed-Meal Tolerance Test (Appendix 2 of  "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus")
AUTHORS: Stephen.Gitelman , Jeffrey A. Bluestone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is Appendix 2 of  "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-Onset Type 1 Diabetes Mellitus".</div><div class = "text-block">This clinical study is supported by JDRF. The aim of the collection is to determine whether imatinib will slow the progression of the autoimmune destruction of ß cells and lead to the preservation of C-peptide secretion in T1DM and to assess Diabetes-related objectives and safety of Imatinib in participants with new-onset type 1 diabetes mellitus".</div></div>

[STEPS]
TITLE: PROTAC GMP Services
AUTHORS: boc.protac  

[DESCRIPTION]
BOC Sciences’ GMP production sites were designed in strict accordance to US-FDA guidelines for GMP Manufacturing. It has passed US-FDA inspections. We provide standard QC tests before releasing GMP products. For GMP products, we guarantee >98% purity. In addition, extra tests can be provided according to the requests of clients. Our GMP manufacturing capabilities are designed to meet actual and future customer needs in respect of all stages of PROTAC drug discovery.

Advantages
Strategic variety encompassed in the combinations of linkers and ligands aids the design of target degraders
State-of-the-art techniques and software tools for ligand design and screening
Standard QC tests before releasing GMP products
Industry-leading turnaround times on deliveries worldwide-and highly competitive pricing
Numerous purification options, such as liquid chromatography isolation, silica gel purification, and high-performance liquid chromatography
Both research-grade or GMP-grade ligand and linker can be customized.
cGMP Manufacturing that is fully traceable

[STEPS]
TITLE: Simpli ACV Keto Gummies Reviews: Fraud Attention, Is Simply Health safe or Scam? Where to Buy!!
AUTHORS: alexhalex  

[DESCRIPTION]
Simpli ACV Keto Gummies are the high level weight reduction recipe that is upheld by natural substances to offer regular weight reduction results. The recipe is clinically supported to re-establish the normal cycle in your body to productively consume off the fat stores and tissues.

[STEPS]
TITLE: Extraction of 1-HP derivatives and HPLC preparation
AUTHORS: Muhammad Zaka Asif, Man Shah, Yosef Smadi

[DESCRIPTION]
This protocol describes day 6 of our workflow to grow worms in liquid culture to induce the production of natural products (in this case, 1-HP derivatives). In this protocol, we extract 1-HP and its derivatives and then prepare the sample for HPLC injection. 

[STEPS]
1. Remove the sample from the lyophilizer. It should appear to be a powder.
2. Resuspend in minimal methanol (enough to submerge the pellet)
3. Vortex the tube with methanol to aid in dissolution.
4. The HPLC injection sample caps out at 90 uL, so minimizing the solution volume here will minimize the total number of injections needed.
5. Transfer the solution to a 2 mL microcentrifuge tube. Centrifuge at 20,800 RCF for 30 minutes.
6. Transfer only the supernatant into HPLC tubes (leave out the solids if any at the bottom of the microcentrifuge tubes). Keep track of the volume so that the injection volume for HPLC will be accurate.
7. Place HPLC tubes in the correct loading sites of the machine and run the protocol.
8. While the HPLC is ongoing, collect fractions if desired (either manually or automatically). Once collected, fractions can be stored at -80˚C for NMR prep later.
TITLE: Laboratory Protocols for Ancient and Modern Dental Calculus DNA Processing (Fellows Yates et al. 2021)
AUTHORS: James Fellows Yates, Irina Velsko, Franziska Aron, Courtney Hofman, Christina Warinner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Collection of protocols used for Fellows Yates </span><span style = "font-style:italic;">et al</span><span>. "The evolution and changing ecology of the hominid primate oral microbiome". Bioinformatics analysis can be found on GitHub at </span><a href="https://github.com/jfy133/Anthropoid_Calculus_Microbiome_Evolution" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/jfy133/Anthropoid_Calculus_Microbiome_Evolution/</span></a><span style = ":;">.</span></div><div class = "text-block">This collection describes the laboratory procedures used for sampling, (ancient) DNA extraction, library construction and preparation for Illumina sequencing of ancient and modern dental calculus samples.</div><div class = "text-block"><span style = "font-weight:bold;">Sampling</span></div><div class = "text-block">- Dental calculus Field-Sampling Protocol (Warinner Version)</div><div class = "text-block">- Dental Calculus Field-Sampling Protocol (Sabin version)</div><div class = "text-block"><span style = "font-weight:bold;">Extraction</span></div><div class = "text-block">- Ancient DNA Extraction from Dental Calculus with Consolidant Removal</div><div class = "text-block">- DNA Extraction from Modern Dental Calculus</div><div class = "text-block"><span style = "font-weight:bold;">Library preparation</span></div><div class = "text-block">- Non-UDG treated double-stranded DNA library preparation for Illumina sequencing of ancient dental calculus </div><div class = "text-block">- (Non-UDG treated) double-stranded modern dental calculus DNA library preparation for Illumina sequencing</div><div class = "text-block"><span style = "font-weight:bold;">-</span><span style = "font-weight:bold;"> </span><span>Full-UDG treated double-stranded ancient DNA library preparation for Illumina sequencing of ancient dental calculus (reduced input DNA)</span></div><div class = "text-block"><span style = "font-weight:bold;">Indexing</span></div><div class = "text-block"><span style = "font-weight:bold;">-</span><span style = "font-weight:bold;"> </span><span>Illumina double-stranded DNA dual indexing for ancient DNA</span></div><div class = "text-block"><span style = "font-weight:bold;">Preparation for Sequencing</span></div><div class = "text-block">- Amplification and Pooling</div></div>

[STEPS]
TITLE: Intravagal AAV injection in mice
AUTHORS: Ayse Ulusoy, Michael Helwig, Angela Rollar, Michael Klinkenberg, Sinead O'Sullivan, Shirley Lee, Rita Pinto-Costa, Donato Di Monte

[DESCRIPTION]
The protocol details AAV vector injections into the mouse vagus nerve and describes pre and post-operative mouse handling.    

[STEPS]
1. Anesthetize the mouse with isoflurane and use pain killers (e.g. Temgesic) following the ethical committee procedures
2. Clean the surgery area with skin disinfectant, make a small incision at the midline of the neck, and
isolate the left vagus nerve. Place a holder underneath to keep it isolated and to form a base to insert the needle.   

3. Using a micropump and a 35-gauge blunt steel needle fitted to a 10-ul NanoFil syringe, inject 800 nl AAV-containing solution. Keep the needle in place for another minute to avoid backflow. 



4. Suture and clean the wound and return the mouse to a clean cage. Follow the ethical committee's recommendations for post-operative care. 

SECTION: post-operative drug treatments
5. Post-operative drug treatments: 


SECTION: post-operative drug treatments
5.1. To stimulate DREADDs, inject 1 mg/kg CNO (Tocris) dissolved in 0.9% saline intraperitoneally.
SECTION: post-operative drug treatments
5.2. For in situ oxidative stress assessments dissolve DHE (Dihydroethidium) in saline/dimethyl sulfoxide (DMSO) (1:1 ratio) and inject intraperitoneally at a dose of 15 mg/kg.