eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Previously, <em>Picelli et al</em> (Picelli et al., 2014) reported a Tn5 transposase-based library construction procedure for Illumina sequencing. Here, we describe an optimised procedure for high throughput library preparation to facilitate large-scale sequencing that does not rely on advanced lab equipment. The Tn-5 transposase used can be purified using a publicly available construct (Picelli et al., 2014). Reaction buffers and primers can be prepared using standard chemicals available from common suppliers.</p>

[GUIDELINES]
<ol>
<li><strong> Introduction</strong></li>
</ol>
<p><strong> </strong></p>
<p>Previously, <em>Picelli et al</em> (Picelli et al., 2014) reported a Tn5 transposase-based library construction procedure for Illumina sequencing. Here, we describe an optimised procedure for high throughput library preparation to facilitate large-scale sequencing that does not rely on advanced lab equipment. The Tn-5 transposase used can be purified using a publicly available construct (Picelli et al., 2014). Reaction buffers and primers can be prepared using standard chemicals available from common suppliers.</p>
<p> </p>
<ol start="2">
<li><strong> DNA input recommendations</strong></li>
</ol>
<p><strong> </strong></p>
<p>2.1. Sensitivity to DNA preparation protocol</p>
<p> </p>
<p>We have tested this protocol with DNA prepared using several different methods (QIAGEN Maxi Blood kits, QIAGEN DNeasy Blood &amp; Tissue Kits, QIAGEN Gentra Puregene Blood Kit) with DNA eluded in water and TE. We did not find the protocol to be sensitive to these factors.</p>
<p> </p>
<p>2.2. DNA quality and quantity</p>
<p> </p>
<p>The quality of the input DNA is not a major concern for preparation of sequencing libraries using this protocol. We have prepared libraries using DNA stored in -20°C for up to ~20 years from chicken and foxes with good result.</p>
<p> </p>
<p>This protocol is optimised for DNA input of 10 ng. Lower DNA input, 1-5 ng, will yield sufficient amount of library for sequencing by increasing the number of PCR cycles. If possible, we would recommend using 10 ng in order to reduce the amount of PCR duplicates.</p>

[STEPS]
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TITLE: JAX-Sen: Collection and shipment of specimen for single-nuclei RNA sequencing (snRNA-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-nuclei RNA-sequencing (snRNA-seq).

[STEPS]
SECTION: Reagents and Materials
1. 2mL Cryovials/ screw-cap tubes
ice cold 1X PBS
Kimwipes
Petri dishes
Liquid Nitrogen
Dry ice
Tweezers (clean, sterile)
SECTION: Quality Key Points:
2. The tissue specimen should be kept at 4 degrees Celsius and RNase-free until snap-freezing and maintained at -80 degrees C or on dry ice thereafter, until it is processed at JAX. 
It is crucial to not store the tissue specimen at RT to avoid freeze-thaw cycles, any cell death, and 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. Animal was euthanized via cervical dislocation. 
SECTION: Procedure:
4.2. Animal was pinned to a necropsy tray which remained on wet ice throughout the harvest.
SECTION: Procedure:
4.3.  Animal was perfused with 20ml cold PBS.
SECTION: Procedure:
4.4. Heart, pancreas, and kidney was collected and the specimen was collected and the specimen was rinsed with ice-cold 1x PBS in a Petri dish to remove blood and other debris.
SECTION: Procedure:
4.5. Pat the specimen dry using clean Kimwipes and freeze as quickly as possible after harvest. 
SECTION: Procedure:
4.6. To flash freeze (snap freeze), place the tissue in a cryo-tube and submerge the tube in liquid nitrogen or immerse deeply in dry ice.
SECTION: Procedure:
4.7. Wait at least 2-3 minutes for the tissue to completely freeze, and transfer the tube containing the tissue to -80 degrees C.
SECTION: Procedure:
4.8. Keep at -80 degrees Celsius thereafter, until shipping.
SECTION: Procedure:
5. Shipment:
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 dry ice (O/N shipping) to:
The Jackson Laboratory for Genomic Medicine,
Farmington, CT, 06032
TITLE: Measuring Fungal Growth
AUTHORS: João Araújo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to measure fungal growth.</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: Protein-coding gene annotation protocol for the eastern banjo frog
AUTHORS: Qiye Li, Qunfei Guo, Yang Zhou, Huishuang Tan, Terry Bertozzi, Yuanzhen Zhu, Ji Li, Stephen Donnellan, Guojie Zhang

[DESCRIPTION]
This pipeline is the protein-coding gene annotation by using homology-based and de novo  predictions to build gene models for the Limnodynastes dumerilii dumerilii genome assembly.

[STEPS]
SECTION: Gene models combination
4. Combine gene models dervied from the above two methods into a non-redundant gene set using a similar strategy to Xiong et al. (2016).

 

SECTION: Gene filtration
5. Remove genes showing BLASTP (blast-2.2.26) hits to transposon proteins in the UniProtKB/Swiss-Prot database (v2019_11), or with more than 70% of their coding regions overlapping repetitive sequences from the combined gene set.

 

SECTION: Functional annotation
6. Finally, perform functional annotation by searching the Limnodynastes dumerilii dumerilii proteins against public databases of UniProtKB/Swiss-Prot (v2019_11), NCBI nr (v20191030), and KEGG (v93.0) with BLASTP (blast-2.2.26;).

 

SECTION: Gene prediction preparation
1. Download protein sequences of diverse vertebrate species (Danio rerio (Ensembl, release-98), Xenopus tropicalis (Ensembl, release-98), Xenopus laevis (NCBI, GCF_001663975.1), Nanorana parkeri (NCBI, GCF_000935625.1), Microcaecilia unicolor (NCBI, GCF_901765095.1), Rhinatrema bivittatum (NCBI, GCF_901001135.1), Anolis carolinensis (Ensembl, release-98), Gallus gallus (Ensembl, release-98) and Homo sapiens (Ensembl, release-98)).
SECTION: Homology-based prediction
2. Align the protein sequences from the previous step to the Limnodynastes dumerilii dumerilii genome assembly by using TBLASTN (blast-2.2.26).


Extract the genomic sequences of the candidate loci together with 5 kb flanking sequences. 


Align the homologous proteins to the extracted genomics sequences from the previous step by using GeneWise (wise-2.2.0) for exon-intron structure determinations.



SECTION: de novo prediction
3. Randomly pick 1,000 homology-derived gene models of Limnodynastes dumerilii dumerilii  with complete open reading frames (ORFS) and reciprocal aligning rates exceeding 90% against the X. tropicalis proteins to train AUGUSTUS (v3.3.1, RRID:SCR_008417).


Use the obtained gene parameters by AUGUSTUS to predict protein-coding genes on the repeat-masked Limnodynastes dumerilii dumerilii genome assembly.
TITLE: Hypotheses, Objectives, Endpoints, and Case Definitions (Part 2 of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is Part 2 of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: Western Blot
AUTHORS: Elena Coccia, gustavo.parfitt

[DESCRIPTION]
Western Blot Protocol

[STEPS]
SECTION: Cell lysis preparation
1. Prepare the appropriate amount of RIPA buffer with Protease and Phosphatases Inhibitor Cocktail (Roche) and maintain on ice

SECTION: Cell lysis preparation
2. Collect cells/organoids in PBS on ice  in an eppendorf and spin down 500 rpm, 5 min, 4 °C 

SECTION: Cell lysis preparation
3. Add appopriate amount of lysis buffers (1:50 v/v proportion between cell pellet and lysis buffer) to the pellet and pipette up and down
SECTION: Cell lysis preparation
3.1. For organoid samples use an homogeniser 
SECTION: Cell lysis preparation
4. Incubate samples for 30 min  on ice . Spinning every 10 min
SECTION: Cell lysis preparation
5. Centrifuge at 15000 x g for 10 min at4 °Cand collect the supernatant in a new tube.

SECTION: Cell lysis preparation
6. If SDS fraction is desired, add 2 Mass / % volume SDS to RIPA buffer and resuspend pellet from step 5.

SECTION: Cell lysis preparation
6.1. Sonicate the sample a 3 times for10 s to ensure fragmentation of genomic DNA released during lysis and consequently reduce viscosity

SECTION: Cell lysis preparation
7. Quantify protein concentration. 
SECTION: SDS-PAGE
8. Mix 20-30 µg of protein with Laemmli's loading buffer

SECTION: SDS-PAGE
9. Denaturate the proteins by incubation at 95 °C for 5 min , spin briefly before loading

SECTION: SDS-PAGE
10. Load pre-cast gel into Western Bloat apparatus and fill with Running Buffer (BioRad).
SECTION: SDS-PAGE
11. Load samples and protein ladder into gels, Run the gel at 120V for 45 min to ensure protein separation.

SECTION: Protein Immunodetection
12. Transfer proteins into a PVDF membrane (previously activated with methanol and hydrated in ddH2O)
SECTION: Protein Immunodetection
13. Remove membrane from transfer and place into a box with blocking buffer: 5% BSA In TBS-T (20mM Tris-HCl, 150mM NaCL pH8, 0.1% Tween20). Block for 60 min

SECTION: Protein Immunodetection
13.1. If alpha-synuclein is to be blotted, fix the membrane with 4% PFA in PBS for 30 min prior to blocking

SECTION: Protein Immunodetection
14. Once blocked, sequentially probe the membrane for antibody staining and detection. Antibody dilution might need optimization.
SECTION: Protein Immunodetection
14.1. Prepare primary antibody in 5% BSA in TBS-T and left incubating 30 min at 4 °C 

SECTION: Protein Immunodetection
14.2. Wash membranes 3x 10 min in TBS-T and then incubated for 60 min at Room temperature with the appropriate HPR-tagged secondary antibody.

14.3. Membrane was washed 3x 10 min in TBS-T
15. Mix EZ-ECL solution 1:1 (v/v) and incubate on the membrane for 1 min 

16. Image membranes using a Licor Odyssey Chemioluminescence Imager.
TITLE: Quick Decapitation for Paraffin Embedding
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to quickly decapitate mice and isolate their brain for paraffin embedding and microtome sectioning.

[STEPS]
1. Sedate mice with isoflurane then quickly decapitate.
2. Extract the brain and incubate in 4% paraformaldehyde or 10% formalin for 72 hours
3. Incubate brain in 70% ethanol and submit for paraffin embedding. Once embedded, section brains at 5μm.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This brief protocol shows you how easy it is to get shRNA from demostrated siRNA sequence. With these self-designed shRNA, I've got more than 10 genes knockdown from mammalian cell lines, including mouse embryonic stem cells. Believe it or not, using siRNA sequence from Dharmacon will guarantee you the success.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol outlines the quality control analysis measuring the percent relative abundance of 16S rRNA levels in the virome and whole metagenome, as well as the number of virome sequences mapping to the whole metagenome, and vice versa. These measures are used to validate the quality of the sequence datasets. Based on the 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.<br /><br /><br />

[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: Maps
AUTHORS: Dakota Betz

[DESCRIPTION]
Basic MarMap instructions for plotting maps in R studio.

[STEPS]
SECTION: Programs and Dependencies
1. 
SECTION: Programs and Dependencies
2. Once you have R and R Studio installed, install marmap
SECTION: Programs and Dependencies
3. Marmap depends on some other packages, and you may encounter errors if they're not up to date. 
SECTION: Troubleshooting Errors
8. Message: 
Error in if (ncol(x) == 3 & !exists("bathy", inherits = FALSE)) { : argument is of length zero
This error occurs when the packages rgdal and raster are not up to date. Update them using

 

SECTION: Getting Started-Quick Maps
6. To start with, you'll want to make some quick/rough maps to make sure you've got your desired geographic range, etc. To make a map, R will first have to query the NOAA database for data for a given geographic range that you provide. Visualizing this by creating maps is really helpful for deciding on the exact range that you want, but you don't want to waste time creating high-quality maps at this stage.

To query the NOAA database, use the following code:
 
To see a quick summary of your plot and generate a quick map in black and white:

 

SECTION: Programs and Dependencies
4. 
SECTION: Getting Started-Quick Maps
5. Before making any maps, be sure that you have the marmap package loaded. Run the following code:

 

SECTION: Color Maps
7. This is my favorite color scheme:
TITLE: Ikaria Lean Belly Juice Reviews (UPDATED) HONEST REVIEW - Is It Best #Supplement?
AUTHORS: health  , health  

[DESCRIPTION]
If you are looking for a natural and healthy way to lose weight, then you should try Ikaria Lean Belly Juice Reviews. This is a revolutionary product that has the ability to help people lose weight without having to go through any strenuous exercise or change their diet.

Ikaria Lean Belly Juice Reviews - Click Here

[STEPS]
TITLE: ELISA for quantification of IL-10 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukin-10 is a pleiotropic cytokine playing a critical role as a regulator of myeloid and lymphoid cell function. Due to the ability of IL-10 to blocking cytokine synthesis and many accessory cell functions of antigen-presenting cells such as macrophages this cytokine is a potent suppressor of the effector functions of macrophages, T-cells and NK cells. IL-10 also participates in regulating proliferation and differentiation of B-lymphocytes, mast cells and thymocytes [1]. </div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Ouyang W, O'Garra A. IL-10 Family Cytokines IL-10 and IL-22: from Basic Science to Clinical Translation.</span><span style = "font-style:italic;">Immunity</span><span>. 2019;50(4):871-891. doi:10.1016/j.immuni.2019.03.020</span></div></div>

[STEPS]
?. An anti-human IL-10 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl  of human serum. Human IL-10 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wasth o remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-10 antibody is added.
?. The microplate is rewashed with PBS-Tween buffer.
?. One hundred µl of streptavidin-HRP is added and binds to the biotin-conjugated anti-human IL-10 antibody.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 50 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-10 present in the sample or standard.
?. The reaction is terminated by addition of 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-10 standard dilutions and the human IL-10 sample concentration determined.
?. For better results place the microplate on a shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol aims at building a methodological synthesis to understand cancerous cells not only by bio lab’s facilities but also by new-media arts lab’s tools, applications, and vision. The template illustrates potential technical, conceptual, and aesthetic inputs derived from the new-media arts lab. On the other hand, three output channels have been emphasized to contribute to the core of the stem cells research, public engagement, and the core of the research in new-media arts.</p>

[STEPS]
TITLE: Overview of difficult airway management cllinical practice guidelines in anesthesia. 
AUTHORS: Ciapponi Agustín, Lucas Perelli, Hernan Cohen Arazi, Germán Solioz, Bardach Ariel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Summary</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Background</span><span>: The aim of the clinical practice guidelines (CPGs) in the management of difficult airway is to provide a structured response to a potentially life-threatening clinical problem. However, there is concern about the extent to which they are based on sound evidence.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Objective</span><span>: Our aim is to summarize and compare relevant recommendations and algorithms from evidence-based CPGs (EB-CPGs).</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"><span style = "font-weight:bold;">Methods</span><span>: We will conduct a systematic review (overview) of CPGs, following Cochrane methods. We will summarize recommendations, its supporting evidence and strength of recommendations according to the GRADE methodology.</span></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">We will search CPGs that were published in the last 10 years, without language restrictions, in electronic databases, and also searched specific CPG sources, reference lists and consulted experts. We will search in PubMed, EMBase, Cochrane Library, LILACS, Tripdatabase and additional sources: National Guideline Clearinghouse, NeLH Guidelines Finder, Guía Salud GPCs en España, GAC guidelines, CMA Infobase: CPGs database, New Zealand Guidelines, Scottish Clinical Guidelines, EBM Guidelines, Health Services/Technology Assessment Text (HSTAT), National Institute for Health and Clinical Excellence (NICE) and Institute for Clinical Systems Improvement (ICSI). Pairs of independent reviewers will select EB-CPGs and rate their methodological quality using the AGREE-II instrument.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">We will include those EB-CPGs reporting standard methods for identification, data collection, study risk of bias assessment and recommendations’ level of evidence. Discrepancies will be resolved by consensus. </div></div></div>

[STEPS]
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