eamag/protocols-with-steps huggingface dataset:
TITLE: Ovarian Tissue Procurement from Organ Donor
AUTHORS: pooja.devrukhkar, hannah.anvari, asma.giornazi Asma Giornazi, shriya.shah, maryellen.pavone, francesca.e.duncan francesca.duncan

[DESCRIPTION]
The purpose of this protocol is to describe the procurement of human ovarian tissue from organ donors for research purposes.

[GUIDELINES]
Researchers will adhere to all safety and training protocols
required including but not limited to: 
1.Biosafety Certification 
2.Bloodborne Pathogens Certification 
3.Collaborative Institutional Training Initiative (CITI program) certification 

[STEPS]
SECTION: Collection of ovarian tissue for research - Done by Pathology
1. The NU-RTL is an established IRB-approved central repository that allows the collection of clinical specimens from participants including but not limited to gynecologic tissue, follicular fluid, and associated bio-fluids.  This material is obtained at the time of surgery and/or clinical procedure and would otherwise be discarded.
SECTION: Collection of ovarian tissue for research - Done by Pathology
2. Reproductive specimens are placed in saline after tissue resection. The amount of tissue, the sites from which it is obtained, and the amount dedicated to the research study are at the discretion of the Attending Physician and the Department of Pathology.
SECTION: Collection of ovarian tissue for research - Done by Pathology
3. The samples are transported fresh and on ice (if necessary) to the Reproductive Tissue Library Core for further processing by trained research staff.
SECTION: Collection of ovarian tissue for research - Done by Pathology
4. Ovaries are evaluated grossly per standard protocol in Northwestern Medicine Pathology. Ovaries are measured, external surface is examined and evaluated for neoplasia.  
SECTION: Collection of ovarian tissue for research - Done by Pathology
5. The ovaries are sectioned in 3-5 mm sections perpendicular to the long axis to evaluate the cut surface (Figure 1).  
 
SECTION: Collection of ovarian tissue for research - Done by Pathology
6. If no significant gross pathology is found, any ovary sections that are not required for clinical diagnosis are handed to the research coordinator. 
SECTION: Collection of ovarian tissue for research - Done by Pathology
7. If gross lesions are found, any non-lesioned tissue based on individual assessment is provided. The sign-out pathologist or study pathologist is consulted in case of questions on what is appropriate to submit or retain.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
Prepare 4mg/mL collagenase solution before start and place on 37C shaker to dissolve.

[STEPS]
SECTION: Prepare materials
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SECTION: Initial Tissue Preperation
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SECTION: Mechanical Dissociation of Tissue
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SECTION: Enzymatic Dissociation of Tissue
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SECTION: Organoid Collection
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SECTION: Single Cell Preperation
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SECTION: Single Cell FACS Preperation
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A sequence-independent method to turn genomic DNA into libraries of hairpins. </p>
<p> </p>
<p>An efficient way to get a different adapter on each end of a DNA fragment is to clone it and digest the fragment back out. Design the vector to have unique restriction sites outside the unique regions you want to put on each end. After digestion, your DNA fragments will have two different "adapters" on each end.</p>
<p> </p>
<p>Adapted from: </p>
<p style="margin-left: 24pt; text-indent: -24.0pt;">Wang L., Zheng J., Luo Y., Xu T., Zhang Q., Zhang L., Xu M., Wan J., Wang MB., Zhang C., Fan Y. 2013. Construction of a genomewide RNAi mutant library in rice. <em>Plant Biotechnology Journal</em> 11:997–1005. DOI: 10.1111/pbi.12093.</p>

[BEFORE_START]
<p>Overview of the procedure:</p>
<ol>
<li>Preparation of DNA fragments (in this example we'll use a fragment of YFP DNA, but this could be random sheared, end repaired DNA, too) (A-tail and kinase to add phosphate).</li>
<li>Prepare vector to clone DNA fragment (T-tail)</li>
<li>Ligate vector and insert (T-A cloning)</li>
<li>Restriction digest to get fragments with unique ends </li>
<li>'Bell' adapter ligation to make the fragment circular. Unique ends on the DNA fragment allow different adapters to ligate to each side.</li>
<li>Rolling circle amplification</li>
<li>Restriction digestion to get single hairpin fragments (inverted repeats of the initial sequence). This can then be cloned and used in whatever downstream application is needed (e.g. RNAi, etc.)</li>
</ol>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/jc8dubw.png" data-src="https://s3.amazonaws.com/pr-journal/jc8dubw.png" data-ofn="Slide1.png" /></p>

[STEPS]
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TITLE: Donor Acceptance Criteria for TMC Florida/Zurich HuBMAP Inclusion
AUTHORS: Marda   Jorgensen, Clive  Wasserfall

[DESCRIPTION]
This document outlines the required criteria for inclusion for donors of thymus, lymph nodes and spleen in the HuBMAP project.

[STEPS]
SECTION: Donor Criteria
1. A copy of the complete recovery checklist must be provided
SECTION: Donor Criteria
2. BMI must be in keeping with CDC published growth charts 5th to 95th percentile
SECTION: Donor Criteria
3. Donor must be less than seven days on ventilator

SECTION: Donor Criteria
4. Donor tissue must be received within 24 hours of recovery

SECTION: Donor Criteria
5. Contraindications for donor acceptance are
SECTION: Donor Criteria
5.1. More than 4 blood transfusions
SECTION: Donor Criteria
5.2. Any autoimmune diseases
SECTION: Donor Criteria
5.3. Chromosomal abnormalities
SECTION: Donor Criteria
5.4. Bacterial or viral meningitis or MRSA in any culture
SECTION: Donor Criteria
5.5. Sepsis
SECTION: Donor Criteria
5.6. Observable infarction, rupture, deflation or gross abnormality
SECTION: Spleen Specific Criterion
6. Must receive at least 50% of the intact spleen, including hilum.
SECTION: Spleen Specific Criterion
7. No spenomegly
SECTION: Lymph Node Specifc Criterion
8. Must receive at least 4 intact lymph nodes
SECTION: Thymus Specific Criterion
9. Must be under the age of 50 to permit for adequate lymphoid tissue presence
SECTION: Thymus Specific Criterion
9. 
SECTION: Donor Criteria
5.7. Immune deficiency diseases
SECTION: Donor Criteria
5.8.  Cancer
SECTION: Donor Criteria
5.9. Any active known infections including SARS-Cov-2
TITLE: Standard Operating Procedure: Microbank
AUTHORS: Romina Gazis-Seregina, Pedro Pablo Parra

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to store and retrieve bacterial and fungal cultures with Microbank™.</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: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes antigen stimulation of human peripheral blood mononuclear cells (PBMC), staining for various surface and intracellular molecular markers, followed by flow cytometry measurements. PBMC contain various T and NK cell populations, which produce cytokines upon specific stimulation with antigens. T (and NK) cells can be stimulated in various ways: using whole virus, bacteria (or membrane fractions of bacterial lysates), antigenic peptides etc. The stimulation guide provided with this protocol refers to stimulation with antigenic peptides, influenza virus and a standard cell mitogen (Concanavalin A, ConA), though it can be adapted to incorporate any other cell stimulant.</p>
<p>This method is used to stimulate influenza specific cytokine production. After PBMC stimulation, cells are stained to detect cytokine producing T (and/or NK) cells by flow cytometry. Antigens are presented by antigen presenting cells (APC), which are also present among PBMC. In this procedure the Golgi apparatus is blocked by Brefeldin A and Monensin in order to keep all the cytokines produced after stimulation within the cell. The number of influenza specific cytokine producing T (and/or NK) cells can be determined by staining cells with fluorophore conjugated antibodies specific for the cytokine(s) of interest and counted on a flow cytometer.</p>

[STEPS]
TITLE: Reconstituting LRRK2RCKW on Microtubules for cryo-EM studies
AUTHORS: Mariusz Matyszewski

[DESCRIPTION]
This protocol contains a short instruction for reconstituting LRRK2RCKW on microtubules for cryo-EM studies with or without kinase inhibitors present.

[BEFORE_START]
Please take notice of the buffer preparation in section 'Materials'.

[GUIDELINES]
Originally used without kinase inhibitors in:
 
Kinase inhibitor step was added for "Structural basis for Parkinson’s Disease-linked LRRK2’s binding to microtubules" by Snead, Matyszewski, Dickey et al.

[STEPS]
SECTION: Instruction
1. Add purified LRRK2RCKW and unpolymerized bovine tubulin dimer in a 2:1 molar ratio into the polymerization buffer (2 LRRK2RCKWs for each tubulin dimer). (Recommended total size: 10 µL) 
 

 

SECTION: Instruction
2. Allow the mixture to polymerize at Room temperature for at least 60 min.
SECTION: Instruction
3. Prepare cryo-EM grids. 
Recommended grids to use: Lacey Carbon on copper, 300 mesh, made by EMS. Glow discharged right before plunge freezing for 45 s  at 20 mA current.

SECTION: Instruction
4. Dilute the sample 3-fold in the LRRK2 reaction buffer. (Recommended mixture: 4 µL + 8 µL).

 

SECTION: Instruction
5. Apply diluted sample to grid and plunge freeze using your usual Vitrobot settings. (Ex. 4 µL, blotted for 4 s at blot force 20 for our particular Vitrobot (FEI); conditions might vary from one machine to another).
SECTION: Instruction
1.1. If incubating with LRRK2 kinase inhibitors, add those before adding tubulin to LRRK2RCKW and allow to incubate for at least 5 min.
TITLE: Cas9 Protein Purification Protocol
AUTHORS: Shu Huang

[DESCRIPTION]
This protocol outlines the steps for purifying Cas9 protein, essential for the preparation of RNP complexes used in CRISPR-Cas9 editing processes.

[STEPS]
SECTION: Expression of Cas9 protein in a suitable host system
1. Express the Cas9 protein in a host system that supports optimal protein yield and stability.
SECTION: Lysis and purification of Cas9 protein
2. Lyse the host cells and purify the Cas9 protein using standard biochemical techniques to ensure high purity and activity.
SECTION: Quality assessment of purified protein
3. Assess the quality of the purified Cas9 protein using appropriate assays to verify its activity and suitability for further applications.
TITLE: Measuring Photophysiology of Attached Stages of Colacium sp. by a Cuvette-Type Fast Repetition Rate Fluorometer
AUTHORS: Takehiro Kazama, Kazuhide Hayakawa, Koichi Shimotori, Akio Imai

[DESCRIPTION]
Fast repetition rate fluorometer (FRRf) is a beneficial method for measuring photosystem II (PSII) photophysiology and primary productivity. Although FRRf can measure PSII absorption cross section (σPSII), maximum photochemical efficiency (Fv/Fm), effective photochemical efficiency (Fq′/Fm′), and non-photochemical quenching (NPQNSV) for various eukaryotic algae and cyanobacteria, almost all FRRf studies to date have focused on phytoplankton. Here, we describe how to measure PSII photophysiology of an epizoic alga Colacium sp.Ehrenberg 1834 (Euglenophyta), in its attached stage (attached to zooplankton) using cuvette-type FRRf. First, we estimated the effects of substrate zooplankton (Scapholeberis mucronata O.F. Müller 1776, Cladocera, Daphniidae) on background fluorescence and σPSII, Fv/Fm, Fq′/Fm′, and NPQNSV of planktonic Colacium sp. To validate our methodology, we recorded photophysiology measurements of attached Colacium sp. on S. mucronata and compared these results with its planktonic stage. Representative results showed how the protocol can determine effects of Ca and Mn on Colacium sp. photophysiology and identify the various effects of Mn enrichment between attached and planktonic stages. Finally, we discuss the adaptability of this protocol to other periphytic algae.

[STEPS]
TITLE: to export w comments
AUTHORS: Maria Gulyakina

[DESCRIPTION]
qa

[STEPS]
SECTION: s1
1. test
SECTION: s1
2. test 2
SECTION: s1
3. test
SECTION: s2
4. test
SECTION: s2
5. test 2
SECTION: s2
6. test 3
SECTION: s3
7. test
SECTION: s3
8. test 2
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Cell viability can be assessed based on various cellular features and mechanisms. These include cell membrane integrity (detected by cell impermeable dyes or leakage of intracellular lactate dehydrogenase (LDH) activity), monitoring of ATP with bioluminescence assays, determining esterase activity with Calcein-AM or Fluorescein-DA, measuring cellular Redox status with MTT, MTS, WST, or XTT, and detecting the mitochondrial membrane potential with JC-1. Various cell viability assays have been developed for plate readers (monitoring absorbance and luminescence), flow cytometry, and image cytometry (e.g. NucleoCounter® NC-3000TM from ChemoMetec); however, none of these assays have been optimized for near-infrared detection with the Odyssey Imaging System.
 
 
 
 This collection contains protocols to perform the assay with 3 different cell lines:
Saponin-treated A431 Cells
Staurosporine-treated Jurkat Cells
Camptothecin-treated RAW264.7 Cells
  
 
 
 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A method allowing the simultaneous quantitation of 21 amino acids in dry leaf tissue using liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) in multiple reaction monitoring (MRM) mode.</p>

[BEFORE_START]
<p>Samples, standards and solvents need to be freshly prepared.</p>
<p>Only use water and solvents of UHPLC-MS/MS grade.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="display: inline !important; float: none; background-color: transparent; color: #000000; font-family: Lato,Verdana,Arial,sans-serif; font-size: 14px; font-style: normal; font-variant: normal; font-weight: 400; letter-spacing: normal; line-height: 20px; orphans: 2; text-align: left; text-decoration: none; text-indent: 0px; text-transform: none; -webkit-text-stroke-width: 0px; white-space: normal; word-spacing: 0px;">Thoracentesis is a procedure in which a needle is inserted into the pleural space between the lungs and the chest wall. This procedure is done to remove excess fluid from the pleural space or to determine the cause of the pleural effusion. The aim of this protocol is to spread to the cientific community the standard procedures to perform thoracentesis and pleural biopsy at HUPE / UERJ</span>.</p>
<p> </p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol gives a method for isolating temperate phages from marine viral concentrations. This protocol uses the conventional plaque agar overlay and looking for turbid or haloed plaques, a hallmark of temperate phages.<br /><br />Paul, J. H., and M. Weinbauer. 2010. Detection of lysogeny in marine environments, p. 30–33. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_030.pdf" target="_blank">full chapter</a> for additional details.

[GUIDELINES]
<strong>Overview<br /></strong>The isolation of temperate phages requires a cultivatable host and a source of concentrated viruses. For example, we isolated a pseudotemperate phage (φHSIC) and its host from the same bacterial/viral concentrate (Jiang et al. 1998) that we obtained in the Sand Island Channel, Oahu, HI, USA. The host was first isolated by standard isolation streaking on marine agar using inoculum from a microbial population concentrate. This protocol uses the conventional plaque agar overlay and looking for turbid or haloed plaques, a hallmark of temperate phages.<strong><br /><br />Materials</strong> <br />
<ul>
<li>Standard marine agar (1.5%) plates (i.e., Zobell 2216 or ASWJP);</li>
<li>Sterile marine broth; </li>
<li>Sterile marine soft agar (1%), 3 mL per 15-mL tube; </li>
<li>Water bath;  </li>
<li>Marine host bacterial culture in exponential growth, 20–50 mL; </li>
<li>Viral concentrate.</li>
</ul>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div class="page" title="Page 2">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>The Odyssey Fc Imager, with 600 channel capabilities, can image agarose gels stained with popular DNA stains, such as ethidium bromide and SYBR Safe DNA stain, with sub-nanogram sensitivity. The Odyssey Fc Imager contains a 532 nm diffuse source with an excitation maximum of 520 nm and a detection maximum of 600 nm. These instrument parameters are within the range of the excitation and emission wavelengths of ethidium bromide (Ex/Em = 302 &amp; 518/605 nm) and other visible fluorescent nucleic acid stains and provide a sensitive gel documentation option.</p>
<p> </p>
<p>Developed for: Odyssey Fc Imaging System</p>
</div>
</div>
</div>
</div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol refers to a study having the aim to identify and test at field artificial structures suitable as collectors for common cuttlefish <em>Sepia officinalis</em> eggs in wild condition.</p>
<p>During the same study, three different protocols were tested and compared. This one refers to the named 'longline' collector protocol, due to their similarity to the longline adopted for fisheries.</p>
<p>This work was supported by the project “Protection, improvement and integrated management of the sea environment and of cross-border natural resources – ECOSEA”, funded by EU Adriatic IPA Cross-border Cooperation 2007-2013 (2°ord./0236/0).</p>
<p> </p>

[BEFORE_START]
<p>An adequate vessel for transshipment/positioning and scuba divers are needed for a proper installation.</p>

[GUIDELINES]
<p>This linear-shaped structure was specifically designed to collect cuttlefish eggs inside longline mussel farms. The longline collector consisted of a 400 m (200 g/m) long lead rope with 250 collectors, represented by 50 cm segments of polyethylene floating rope (diameter 0.8 cm) having a float of 40 g at the free end. Two structures were positioned inside two mussel farms located at South of Ancona  (Italy) concomitantly with the beginning of the spawning period of the cuttlefish.</p>
<p>This work was supported by the project “Protection, improvement and integrated management of the sea environment and of cross-border natural resources – ECOSEA”, funded by EU Adriatic IPA Cross-border Cooperation 2007-2013 (2°ord./0236/0).</p>
<p> </p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/5e52xfe.jpg" data-src="https://s3.amazonaws.com/pr-journal/5e42xfe.jpg" data-ofn="longline_collector.jpg" /></p>
<p> </p>
<p> </p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/5dg2xfe.jpg" data-src="https://s3.amazonaws.com/pr-journal/5df2xfe.jpg" data-ofn="Longline collector.jpg" /></p>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>HBV genotyping was performed by sequencing and phylogenetic analyses of the surface (S) and core (C) fragments. Briefly, HBV DNA was first extracted from 400 µL of plasma and eluted in 100 µL  of pure water, using the QIAamp Viral DNA Mini Kit (QIAGEN, Courtaboeuf, France) followed by semi-nested PCR amplification of the S (930 bp) and C (1010 bp) gene fragments using MP <em>Taq</em> Core Kits 25 (MP Biomedical Diagnostic, Europe). The S fragment amplification was performed as described elsewhere.</p>
<p>(Hu X, et <em>al</em>., 2000 [PMID: 10677515] ; Makuwa M et <em>al</em>., 2006 [PMID: 16847965]; Olinger CM et <em>al</em>., 2006 [PMID: 16603517])</p>
<p>The first round was performed using primers sets 58P (5’-CCT GCT GGT GGC TCC AGT TC-3’) and 979 (5’-ATT GGA AAG TAT GTC AAA GAA TTG TGG GTC TTT TG-3’). The 50 µL final reaction mixture contained 31.4 µL of RNase DNase Free water, 5 µL of buffer 10X with MgCl<sub>2 </sub>(25 mM), 0.4 µL of dNTPs (25 mM), 1.5 µL of each primer (10 µM), 0.2 µL of <em>Taq</em> polymerase (5 U/µL) and 10 µL of extracted DNA. The second round PCR used the 58P/Mc2r (5’-TGGAAGTTGGGGATCATTGCC-3’) primer on a 50 µL final reaction mixture containing 36.4 µL of RNase DNase Free water, 5 µL of buffer 10X with MgCl<sub>2 </sub>(25 mM), 0.4 µL of dNTPs (25 mM), 1.5 µL of each primer (10 µM), 0.2 µL of <em>Taq</em> polymerase (5 U/µL), and 5 µL of first round PCR product. The PCR program was the same for the first and second round PCRs  including denaturation at 94°C for 5 min followed by 40 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 30 s and elongation at 72°C for 1 min, followed by final elongation at 72°C for 5 min.  For the C fragment amplification, the couplet of primers BCP1F (5’-GCA TGG AGA CCA CCG TGA AC-3’) / 2853N (5’-TCA CCA TAT TCT TGG GAA CA-3’) was used for the first round and the couplet BCP2F (5’-CAT AAG AGG ACT CTT GGA CT-3’) / 2853N for the second round. The amplification conditions were the same as for the S fragment.      </p>
<p>The PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Courtaboeuf, France) and submitted for sequencing at Macrogen Inc (Meibergdreef, Netherlands).</p>

[STEPS]
TITLE: Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR
AUTHORS: Wenjing  Liu, Shihong Fu

[DESCRIPTION]
   you can use this protocol to detect the RNA of your sample 

[BEFORE_START]
RNA was extracted from all collected samples (serum, CSF, and mosquito grinding supernatant) using a Tianlong Nucleic Acid Automatic Extractor with the Tianlong Nucleic Acid Extraction Kit(EX-RNA/DNA virus, Suzhou Tianlong Biotechnology Co., Ltd, Suzhou, China).

[STEPS]
SECTION: Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR
1. The instrument used was a Stratagene ™, real time PCR machine (manufacturer: Thermo Scientific, model: MX300) for quantitative fluorescence qRT-PCR detection of JE virus and West Nile virus. AgPath-IDTMOne-step RT-PCR Kit (REF: AM1005, manufacturer: Thermo Fisher Scientific.) was the detection kit used, the total volume of the detection system was 20 μl, containing 10 μl of 2Xbuffer4 μl of RNAse Free Water4 μl, 1 μl of primers, probes, and enzyme mixture (Mix), and 2 μl of template RNA. 
SECTION: Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR
2. The reaction program was: 45 ° C for 10 min, 95 ° C for 10 min for 1 cycle, 95 ° C for 15 s and 60 ° C for 1 min for 40 cycles. Using primers and probes specific for the JE virus gene  and the West Nile virus gene.
SECTION: Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR
3. The final result is checked on the  real time PCR machine,the sample with CT value over 35 thought to be negetive, sample with CT value lower than 35 thought to be positive.
SECTION: Detection of JEV and WNV by quantitative real-time reverse-transcription (qRT)-PCR
4. The positive sample will be sent to do PCR specific to JEV genes to be ferther tested.