eamag/protocols-with-steps huggingface dataset:
TITLE: Preparação para ir ao campo
AUTHORS: Alicia Rafaelly Vilefort Sales, Daniel Vartanian, Maria Augusta Medeiros Andrade

[DESCRIPTION]
Este protocolo foi desenhado para o processo de coleta de dados do projeto de pesquisa Associações entre a duração e a qualidade do sono de gestantes no terceiro trimestre com a duração do trabalho de parto. Consulte a aba Guidelines para visualizar o fluxograma do processo como um todo.

Uma coleção com todos os protocolos do processo deve ser publicada em breve.

Caso tenha dúvidas ou sugestões, entre em contato com um dos autores.

---

This protocol was designed for the data collection process of the research project Associations between the duration and quality of sleep of pregnant women in the third trimester with the duration of labor. See the Guidelines tab to view the process flowchart as a whole.

A collection with all the process protocols should be published soon.

If you have any questions or suggestions, please contact one of the authors.

[BEFORE_START]
Este protocolo assume que o(a) usuário(a) se encontra na sala do Grupo Interdisciplinar de Pesquisa em Sono (GIPSO). Os materiais citados neste documento já devem estar à disposição.

Entre em contato com a supervisão do projeto caso encontre algum problema.

[GUIDELINES]
O processo de coleta de dados deste projeto segue o seguinte fluxograma.

  
 Com exceção da etapa Processe e analise os dados, todas as etapas possuem protocolos próprios.

[STEPS]
SECTION: Agenda
1. Consulte a agenda do projeto e verifique a quantidade de gestantes a serem recrutadas no seu turno.

 

SECTION: Agenda
2. Verifique os horários e o trajeto que você terá que fazer para que você chegue com ao menos 30 minutos de antecedência na Casa de Parto.
SECTION: Actígrafos
3. Separe um actígrafo para cada gestante a ser recrutada e mais um actígrafo extra por segurança.

 

SECTION: Actígrafos
4. Realize o processamento pré-coleta dos actígrafos.

 

SECTION: Actígrafos
5. Armazene os actígrafos de maneira segura.

 

SECTION: Outros materiais
6. Separe os documentos abaixo conforme a quantidade de gestantes que irá recrutar, sempre deixando algumas cópias extras por segurança.

 

Termo de Consentimento Livre e Esclarecido  (TCLE).

 

 Formulário de campo.

 

Termo de entrega de actígrafo.

 

Critério de elegiblidade.
Guia de instruções e cuidados com o actígrafo.
Formulário de controle.

Separe também os seguintes itens conforme a quantidade de gestantes:

Saco plástico hermético de 35 x 45 cm.
Braçadeira de 30 cm.
SECTION: Outros materiais
7. Separe também os seguintes itens:

1 Folha de acesso.
3 canetas esferográficas de cor azul ou preta.
1 Porta crachá.
1 Prancheta.

 

SECTION: Aparelho celular
8. Certifique-se que você tem acesso à internet pela sua operadora de telefonia.

 

SECTION: Aparelho celular
9. Certifique-se que seu celular está com 100% de bateria.

 

SECTION: Ida ao campo
10. Siga para o recrutamento de voluntárias na casa de parto.
TITLE: Platelet purification and coating of plates for adhesion assays 
AUTHORS: Samantha King

[DESCRIPTION]
This is the protocol we use to prepare fixed platelets from human blood and coat wells ready for adhesion assays.  

[GUIDELINES]
We typically have greater success when using at least 180 mls of blood. When using smaller volumes of blood we get a smaller number of platelets than expected

[STEPS]
1. Blood from healthy donors was collected in 1/6th volume of acid citrate dextrose solution (ACD). Mix gently.
2. Blood was spun down for 20 min at 200 x g, deaccelerating step without brake.
3. Platelet rich plasma was collected (top yellow layer) and mixed (inverting the tube) 1:1 with HEP buffer.
4. Platelets were collected by centrifugation at 800 x g for 20 min without brake. At this step supernatant was removed without disturbing the pellet.
5. Platelets were washed twice with 1 ml platelet wash buffer as indicated: rinse the platelet pellet with
platelet wash buffer (without resuspension in order to avoid unnecessary platelet activation) by gently adding wash buffer and removing it slowly with a pipette. Repeat once. After the last wash, resuspend the pellet in 3 ml wash buffer.
6. Platelets were fixed with 1% paraformaldehyde for 10 minutes, incubate platelets at room temperature for 10 minutes.
7. Pellet platelets by centrifuging at 700 x g for 10 minutes (no brake) and wash them with 10 ml PBS pH
7.4 Repeat once.
8. After the second wash, resuspend platelets in PBS pH 7.4 (If we start with 180 mls of blood we typically use 10 mls here).  Count platelets and then adjust the volume of PBS to have 1x107 cells/mL.
9. Coat flat bottom 96-well plate wells with 100 µL of platelets (1x107). We coat triplicate wells for each sample and for each strain coat three control wells with 100 µL of 1% BSA. 
10. Incubate the plate for 1h at 37°C.
11. Aspirate liquid and wash wells by adding 120 µL of PBS to each well. Remove liquid again and finally add 120 µL of PBS to each well.
12. The plates are covered in parafilm and keep in the cold room for further use. They keep at least one month
TITLE: DNA extraction (BOMB)
AUTHORS: Tsu-Chun Hung, Yin-Tse   Huang

[DESCRIPTION]
DNA extraction (BOMB)

[STEPS]
SECTION: Sample Collection
1. Add 200 µL of 1mm beads to 2ml eppendorf tube

SECTION: Sample Collection
2. Add200 µL of 0.5mm beads to 2ml eppendorf tube

SECTION: Sample Collection
3. Add867 µL Lysis master mix to 2ml eppendorf tube

 

SECTION: Sample Collection
4. Collect 10-20 mg of sample to 2ml eppendorf tube
SECTION: Sample crush
5. Put 2ml eppendorf tube in mixmill for sample crush, at this condition: 30 rpm/s, for 4mins 4 min 
SECTION: Centrifugation
6. Put 2ml eppendorf tube in centrifuge for centrifugation, at this condition:17.0 x g, 3 min, 25 °C 
SECTION: DNA purification
7. Add 350 µL of isopropanol to the 1st well of 96 well plate

   
SECTION: DNA purification
7.1. Add 125 µL of magnetic beads (10 mg/ml) to the 1st well of 96 deep well plate 

 


SECTION: DNA purification
7.2. Add 200-300 µL of the sample (lysate) from the 2ml centrifuged tube to the 1st well of 96 deep well plate 
 

SECTION: DNA purification
8. Add 400 µL of isopropanol to the 2nd well of 96 deep well plate 
SECTION: DNA purification
9. Add 300 µL of 80% enthanol to the  3rd well of 96 deep well plate 
SECTION: DNA purification
10. Add 300 µL of 80% enthanol to the 4th well of 96 deep well plate 
SECTION: DNA purification
11. Add 100 µL of DEPC-treated water to the 6th well of 96 deep well plate 
SECTION: DNA purification
12. Put the prepared 96 deep well plate in the automated DNA extraction machine and select the BOMB protocol

SECTION: DNA purification
13. After the extraction is done, collect 50-100 µL of the eluted sample as the DNA template for downstream experiments
TITLE: U Mass - Ammonia
AUTHORS: Jason Kim

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

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Ammonia test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Tissue H&amp;E Staining | HuBMAP | JHU-TMC
AUTHORS: Kyu Sang Han, Pei-Hsun Wu, Joel Sunshine, Ashley Kiemen, Miklhail James, Sashank Reddy, Denis Wirtz

[DESCRIPTION]
The following are guidelines that will have an effective staining window of 2 to 5 minutes. Developed as progressive stains, the intensity of nuclear staining will increase as the time increases. The hematoxylin and eosin stains will have an expected throughput of 2,000-2,500 slides per 500mL bottle. Actual results may vary from lab to lab depending on the staining equipment used, control of carry over into each solution, and length of time stains are left exposed. As a general rule, we recommend changing the hematoxylin and eosin stains once per week if throughput has not been reached. Clarifying and Bluing solutions should be changed more often.

The Leica autostainer is a single fully automated integrated stainer used for standard
H&E staining and operates by progressing each slide through a series of chemical 
changes that first deparaffinize and then stains the tissue slides for histologic review. To 
use, the machine is powered on and the covers are removed from the staining vessels 
every morning and the hematoxylin is filtered daily. The daily number of slides stained is
added to the log sheet beside the stainer, and all the solutions are changed when the 
slide tally reaches 250. At the end of the day, the staining vessels are then covered and
the machine is powered off

[STEPS]
SECTION: H&amp;E staining
17. Final Step: Mount and Coverslip with Covermount
4. Set 2 stations with 100% ethanol. Submerge the slide for a minute in each station.
SECTION: H&amp;E staining
6. Wash the slide in running warm water for a minute
SECTION: H&amp;E staining
7. Submerge the slide in Optik Hematoxylin for 3 minutes
SECTION: H&amp;E staining
11. Submerge the slide in Optik Bluing Solution Type 1 for a minute
SECTION: H&amp;E staining
10. Wash the slide in running warm water for a minute
SECTION: H&amp;E staining
9. Submerge the slide in Optik Aqueous Clarifier Type 1 for a minute
SECTION: H&amp;E staining
8. Wash the slide in running warm water for a minute
SECTION: H&amp;E staining
12. Wash the slide in running warm water for a minute
SECTION: H&amp;E staining
14. Set a station with 95% ethanol. Submerge the slide for a minute in each station.
SECTION: H&amp;E staining
13. Submerge the slide in Optik Eosin Y Type 1 for a minute
SECTION: H&amp;E staining
16. Set 3 stations with Xylene. Submerge the dewaxed unstained slide for a minute in each station.
3. Set 2 stations with Xylene. Submerge the unstained slide for 3 minutes in each station.
SECTION: Before staining
1. Bake unstained slides for 30-60 minutes at 60 degrees Celsius.
SECTION: Dewaxing
2. The following steps require setting up multiple stations with different solutions.
5. Set a station with 95% ethanol. Submerge the slide for a minute in each station.
SECTION: H&amp;E staining
15. Set 2 stations with 100% ethanol. Submerge the slide for a minute in each station.
TITLE: Immunohistochemistry
AUTHORS: Peter Kilfeather

[DESCRIPTION]
This protocol describes the immunohistochemistry protocol.

[GUIDELINES]
This protocol needs prior approval by the users' Institutional Animal Care and Use Committee (IACUC)

[STEPS]
SECTION: Tissue Collection and Storage
4. Dissect and extract the brain.
SECTION: Tissue Collection and Storage
5. Store the brain in 4 % PFA at 4 °C overnight.
SECTION: Dehydration
6. Slice each brain into 3 mm coronal sections (approximately).
SECTION: Dehydration
7. Pass each section through graded alcohol solutions (50, 70, 80, 95, 100, 100 and 100 % ethanol), holding for 10 minutes in each.
SECTION: Dehydration
8. Transfer each section into xylene three times, for 20 minutes each.
SECTION: Dehydration
9. Transfer each section into paraffin two times, for two hours each.
SECTION: Brain Preparation
1. Euthanize the mouse by lethal intraperitoneal injection of pentobarbital.
SECTION: Sectioning
11. Take coronal sections at 7 µm using a Leica microtome (Leica Microsystems, Wetzlar, Germany) and mounted on glass slides (VWR, Superfrost Plus)
SECTION: Brain Preparation
2. Upon loss of the pedal withdrawal reflex, dissect the thoracic cavity open.
SECTION: Brain Preparation
3. Perfuse 25 mL phosphate-buffered saline (PBS) transcardially, followed by 25 mL 4 % paraformaldehyde (PFA), diluted in PBS.
SECTION: Dehydration
10. Embed each section in fresh paraffin, orienting as desired.
SECTION: Deparaffinisation, Antigen Retrieval and Blocking
12. Transfer each slide-mounted set of sections through xylene (twice) and reversed graded alcohols (100, 95, 70, 50 %) for 10 minutes each.
SECTION: Deparaffinisation, Antigen Retrieval and Blocking
13. Transfer to 1X citrate buffer (ab96678, Abcam) and perform antigen retrieval by microwave heat (20 minutes).
SECTION: Deparaffinisation, Antigen Retrieval and Blocking
14. Block for one hour at room temperature using 10 % donkey serum in tris-buffered saline (TBS).
SECTION: Primary Staining
15. Apply primary antibodies to TH (Abcam Cat# ab76442, RRID:AB_1524535, 1:500), GFP (Thermo Fisher Scientific Cat# A-11122, RRID:AB_221569, Invitrogen, 1:1000), CASR (Abcam Cat# ab79038, RRID:AB_2071489, 1:100) or TYROBP/DAP12 (Abcam Cat# ab283679, 1:100), prepared in 1 % donkey serum in TBS and incubate overnight at 4 °C
SECTION: Secondary Staining
16. Wash slides in TBS three times, for five minutes each.
SECTION: Secondary Staining
17. Incubate in secondary antibodies (Alexa Fluor-conjugated anti-rabbit/chicken A-31573,  RRID:AB_2536183 and A78952, RRID:AB_2921074 1:1000) and DAPI (Thermo Fisher Scientific Cat# D3571, RRID:AB_2307445, 1:5000), prepared in TBS for 1 hour at room temperature.
SECTION: Secondary Staining
18. Wash slides in TBS three times, for five minutes each.
SECTION: Secondary Staining
19. Mount coverslips using FluorSave mounting medium (Millipore, Massachusetts, United States of America).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<strong>Viral Bioinformatic Resource Centre</strong>
<ul>
<li><strong>Provide databases of viral genomic information.</strong>
<ul>
<li>Please check the <strong><em>Organisms</em></strong> menu to see which viruses we support: we’re now focusing on large DNA viruses</li>
<li>The VOCs (Virus Orthologous Clusters) database is at the heart of our system.</li>
<li>The database links directly to integrated tools for comparative analyses.</li>
<li>VOCs sorts genes into ortholog clusters (e.g. RNA polymerase) to simplify data retrieval.</li>
</ul>
</li>
<li><strong>Provide easy access to the genes, gene families, and genomes of the different virus families.</strong>
<ul>
<li>via a unique series of powerful Java tools that support multiple computer platforms (see <strong><em>VBRC Tools</em> </strong>menu).</li>
<li>design and build software to tackle specific bioinformatics/virology problems, often in collaboration with virologists.</li>
</ul>
</li>
<li><strong>Rally the research community to provide expert curation of these viral genomes by:</strong>
<ul>
<li>Adding value to GenBank sequences through enhancing and updating genome annotations</li>
<li>Linking to research reviews/papers for the research community.</li>
</ul>
</li>
<li><strong>Collaborate with researchers to help on specific bioinformatics problems, e.g.</strong>
<ul>
<li>Custom searches of the databases</li>
<li>Building new features into our tools</li>
<li>Help with genome annotation</li>
</ul>
</li>
</ul>

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

[DESCRIPTION]
<p>In agro-ecosystems, pollinators are essential for orchard, oilseed crop, horticultural and forage production, as well as the production of seed for many root and fibre crops. Pollinators such as bees, birds and bats affect 35 percent of the world’s crop production, increasing outputs of 87 of the leading food crops worldwide, plus many plant-derived medicines in the world’s pharmacies. Just as the agricultural community is taking stock of the contribution of pollination to crop production, populations of managed pollinators (the Western honey bee Apis mellifera, the Eastern honey bee Apis cerana, and their Asian relatives) are experiencing new and poorly understood threats. Wild pollinators in agricultural landscapes can provide important pollination services and serve also as a critical form of insurance against the risks of pests and diseases amongst managed pollinators. Within the context of its lead role in the implementation of the Initiative for the Conservation and Sustainable Use of Pollinators (also known as the International Pollinators Initiative-IPI) of the United Nations Convention on Biological Diversity adopted in 2000 (COP decision V/5, Section II), FAO has established a “Global Action on Pollination Services for Sustainable Agriculture”. FAO has also developed a global project, supported by the Global Environment Facility (GEF) through the United Nations Environment Programme (UNEP) entitled “Conservation and management of pollinators for sustainable agriculture, through an ecosystem approach”. Seven countries (Brazil, Ghana, India, Kenya, Nepal, Pakistan and South Africa) have worked together with FAO to identify and carry out targeted activities that can address threats to pollinators in agricultural landscapes. The outcomes of the global project are expected to expand global understanding, capacity and awareness of the conservation and sustainable use of pollinators for agriculture. As a contribution to the IPI, FAO and its partners have collaborated with INRA (Institut National de la Recherche Agronomique, a public research body of the French government) to develop a protocol for assessing and detecting if a crop production system is suffering a pollination deficit. Field testing and adaptation of the protocol for the variable cropping systems in different countries was made possible through a grant from the International Fund for Agricultural Development (IFAD) on the “Development of Tools and Methods for Conservation and Management of Pollination Services for Sustainable Agriculture“, in 2009 and 2010. This document thus presents a handbook for the application of the protocol, outlining the underlying concepts, the hypothesis to be tested, and the modification and application of the protocol to a variety of circumstances in developing countries, such as small fields, home gardens, and high environmental variability. As the protocol is applied, FAO and its partners will be able to provide information on the results of detecting and assessing levels of pollination deficit in crops important for nutrition and food security around the world.</p>

[STEPS]
TITLE: LC-MS/MS Label-Free Proteomic Data Acquisition
AUTHORS: Danielle Gutierrez, Jamie Allen, Zach Jenkins, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Description of settings used to acquire LC-MS/MS data from label-free proteomic samples.</div></div>

[STEPS]
?. Label-free proteomic samples were analyzed on a Thermo Scientific Orbitrap Fusion Tribrid mass spectrometer in line with an EvoSep One HPLC system.
?. Samples,1 µL at a 1 µg/µL, were loaded onto EvoTips and diluted with 19 µL of mobile phase A (100% water, 0.1% formic acid). Samples were processed on tip according to the manufacturer's recommended protocol. EvoTips were placed onto the EvoSep One, and samples were eluted from the EvoTips onto a Dr. Maisch C18 AQ, 100 micron inner diameter, 15 cm long column with 3 micron particles (EV1074) and analyzed using the 30 sample per day method settings.
?. Peptides were separated at a flow rate of 500nL/min over a 44 minute gradient according to the 30 samples/day settings. Mobile phase A was 100% water, 0.1% formic acid, and mobile phase B was 100% acetonitrile, 0.1% formic acid.
?. Eluted peptides were ionized via positive mode nanoelectrospray ionization (nESI) using a Nanospray Flex ion source (Thermo Fisher Scientific).
?. The mass spectrometer was operated using a top 12 data-dependent acquisition mode.
?. Fourier transform mass spectra (FTMS) were collected using 60,000 resolving power, an automated gain control (AGC) target of 1e6, and a maximum injection time of 50 ms over the mass range of 375-1500 m/z.
?. Precursor ions were filtered using monoisotopic precursor selection of peptide ions with charge states ranging from 2 to 6. Previously interrogated precursor ions were excluded using a 30 s dynamic window (± 10 ppm).
?. Precursor ions for tandem mass spectrometry (MS/MS) analysis were isolated using a 1.2 m/z quadrupole mass filter window and then fragmented in the ion-routing multipole via higher energy dissociation (HCD) using a normalized collision energy of 35%.
?. Ion trap fragmentation spectra were acquired using an AGC target of 10,000 and maximum injection time of 60 ms, and 120 m/z was set for the first scan mass to enable detection of the lysine residue fragmented ion.
TITLE: Assembling of  Synoecnema hirsutum  mitochondrial genome
AUTHORS: Elena  S Ivanova, Boris  D. Efeykin,   Sergei E Spiridonov

[DESCRIPTION]
Synoecnemahirsutum Timm, 1959 (Ungellidae, Drilonematoidea) found in the body cavity of the pheretimoid earthworm at the border of Laos and Vietnam was redescribed and illustrated. It was molecularly characterised for the first time. It is also the first member of the superfamily Drilonematoidea for whom the mitochondrial genome was obtained.

[STEPS]
1. DNA from the frozen nematodes samples was isolated using the QiAmp Micro Kit (Qiagen) according to a standard protocol.
2. DNA library preparation was implemented using the NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA).
3. DNA quality was checked with Qubit 3.0, final library length distribution and checking for the absence of adapters was performed using Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA).
4. Sequencing was performed on Illumina HiSeq system with a 150 bp read length at the Skoltech Genomics Core Facility (https://www.skoltech.ru/research/en/ shared-resources/gcf-2/).
5. The quality of raw reads was evaluated using FastQC (Andrews 2010).
6. After filtering by quality, remaining readings were cleared by removing of cDNA synthesis adapters and sequence adapters by processing with the Trimmomatic program.
7. De novo assembly was implemented using Velvet (Zerbino et al. 2010) with the default settings.
8. The resulting contigs were filtered by length, and contigs with the most similarity in size to mitochondrial DNA were selected. Assembled sequences of protein-coding genes were checked for internal stops in PCGs manually.
9. The contigs were annotated using the MITOS web server (Bernt et al. 2013), with the default settings.
10. Prediction of protein-coding genes and rRNA genes was done by using a combination of BLAST and MITOS online software. Concatenated nucleotide alignment of 13 protein-coding genes was performed using Geneious Prime 2019.1 (Biomatters Ltd., Auckland, New Zealand).
11. Conserved regions in the alignments of the 13 PCGs were selected using the GUIDANCE2 server (Sela et al. 2015).
12. Optimal evolutionary models were chosen by PartitionFinder (Lanfear et al., 2012).
13. Maximum Likelihood (ML) analysis was performed using IQ-TREE web server (Trifinopoulos et al. 2016) with 10,000 ultrafast bootstrap replicates (Hoang et al. 2018).
14. Tree was visualized using TreeView and FigTree v1.4.2 (http://tree.bio.ed.ac.uk/software/figtree/).