eamag/protocols-with-steps huggingface dataset:
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>

[GUIDELINES]
<h3>Â </h3>
<p>Â </p>

[STEPS]
?. 
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TITLE: Análise de metabarcoding de DNA
AUTHORS: Thiago Mafra Batista

[DESCRIPTION]
Este tutorial conduzirá estudantes e pesquisadores a realizarem análise metataxonômica de microrganismos a partir da amplificação das regiões V3V4 do gene 16S ribossomal. Iniciando pela união dos pares de reads com usearch, em seguida, usamos o cutadapt para cortar os primers das sequências. Depois, realizamos a filtragem de qualidade com usearch, encontramos sequências únicas e abundantes, e criamos clusters de OTUs (Operational Taxonomic Units) com 97% de similaridade. Em seguida, utilizamos o mapseq para mapear as OTUs contra um banco de dados. Por fim, criamos tabelas de OTUs para cada amostra e geramos um arquivo HTML para visualização dos resultados com o Krona.

[STEPS]
SECTION: Análise da qualidade das reads
1. Vamos avaliar o perfil de qualidade das reads com o software FastQC.

 

SECTION: União dos pares de reads
2. Cada par de leitura é oriunda de um fragmento de amplicon sequenciado. Este fragmento precisa ser reconstruído. O parâmetro -fastq_mergepairs do usearch faz isso. Troque "{AMOSTRAS}" por um código único de sua escolha.
 

SECTION: Remoção dos primers
3. As sequências dos primers também são sequenciadas e precisamos removê-las da reads, agora, unidas. Vamos utilizar o cutadapt e as sequências dos primers forward (parâmetro -g) e reverse (parâmetro -a).

 

SECTION: Descarte de reads com erros
4. Vamos agora descartar reads que provavelmente possuem erros, tanto nas sequencias que tiveram primers removidos, quanto nas sequencias sem primers identificados.

 

SECTION: Sequências únicas e abundantes
5. Agora vamos encontrar as sequências únicas e abundantes com o parâmetro -fastx_uniques

 

SECTION: Clusters de OTUs
6. Agora vamos agrupar as sequências únicas e abundantes de acordo com a identidade entre elas, considerando 97% de similaridade.

 

SECTION: Tabelas de OTUs
7. Agora vamos criar uma tabela contendo as informações de OTUs:

 

SECTION: Plotar a curva de rarefação da diversidade alfa
8. Vamos analisar a diversidade alfa a partir da curva de rarefação. Para isso vamos utilizar o script rare.R.

 

SECTION: Mapear as OTUs contra um banco de dados
9. Com o mapseq vamos fazer blast das OTUs contra um banco de dados. No exemplo a seguir, utilizamos 24 processadores

 

SECTION: Criar tabelas de OTUs para cada amostra
10. Vamos criar uma tabela que irá conter as informações do mapeamento de cada amostra a partir do resultado do mapseq. Vamos usar um loop para otimizar a execução do awk.

 

SECTION: Concatenar as tabelas geradas com usearch e mapseq
11. Vamos unir as tabelas geradas pelo usearch e pelo mapseq para serem visualizadas no krona. Vamos utilizar o script em perl concat_OTUs_Taxons.pl. 

 

SECTION: Criar o arquivo HTML para visualização dos resultados.
12. 
 

SECTION: Script para otimização da análise
13.
TITLE: Clever Pennies: Honesty Oaths, Misreporting Performance, and MTurk
AUTHORS: J Jobu Babin, Haritima Chauhan, Feng Liu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Workers in online labor markets routinely misrepresent the value of effort, both through shirking and misreporting performance.  We execute a field experiment on Amazon MTurk to  determine  whether  honesty  oaths  impact  the  cost  of  worker  lies.  Employing  a  design that follows the “die roll” paradigm, we determine that oaths elicit more truthful behavior, although they primarily impact extreme lies.  Oath-taking decreased the cost of misreportingby 12%.  While shirking is widespread, oaths marginally decrease it.  Male workers misreport performance more often on average than females, yet both sexes are equally responsive to oath-taking.</div><div class = "text-block">JEL Codes: D90, D91, C8, C93, J9</div></div>

[STEPS]
TITLE: DNA Extraction from Arabidopsis thaliana
AUTHORS: lilyli

[DESCRIPTION]
This is a protocol for extract DNA from arabidopsis thialiana. 

[STEPS]
1. Add 400 mL DNA extraction solution into 1.5 mL centrifuge tube.

2. Grind 3 small leaves or 1 large leaf with a grinding stick in the centrifuge tube with the extract.
3. Shake the oscillator for 5 s, and place it at room temperature until other preparations are completed.

4. Centrifuge at 16 000 rpm for 2 minutes at room temperature.
5. Transfer 200 mL of centrifuged supernatant into a clean centrifuge tube.
6. Add 200 mL isopropanol, mix well and place at room temperature for 3 minutes.
7. Centrifuge at 16 000 rpm at room temperature for 5 minutes, remove the supernatant, wash the precipitate with 70% ethanol, and dry the precipitate at room temperature.
8. Dissolve DNA in 100 mL deionized water and store it in a refrigerator at – 20 ° C.
TITLE: Electrophysiological Recordings
AUTHORS: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol explains Quantitative Real‐Time Polymerase Chain Reaction (qRT‐PCR) of lines ND1014, N1, and ND27760 from </span><span style = "font-style:italic;">Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons</span><span>. </span></div></div>

[STEPS]
?. [Voltage-Clamp Recording]
Perform voltage-clamp recordings at  in a chamber perfused with regular artificial cerebrospinal fluid flowing at 3 ml/minute.Regular artificial cerebrospinal fluid: at  , equilibrated with 95% O2 and 5% CO2, ∼310 mosm)
35 °C
[NaCl]
[KCl]
[MgCl2]
[CaCl2]
[NaH2PO4]
[NaHCO3]
[glucose]
Patch electrodes should be pulled from borosilicate glass and have resistances of 2.0–4.0 MΩ when filled with an intracellular solution. Intracellular solution:   ,        at   , ∼290 mosm
?. [Voltage-Clamp Recording]
Identify neurons using a ×10 objective mounted on an upright microscope with transmitted light.
?. [Voltage-Clamp Recording]
Cell somatic recordings should be made using an Axopatch 200B amplifier in combination with pClamp 10.7 software (Molecular Devices).
Voltage clamp at −70 mV.Monitor Rseriesand Rinput using a 2.5‐mV 100‐ms depolarizing voltage step in each recording sweep.Filter current traces at 5 kHz, digitize at 10 kHz using a Digidata 1440 interface, and store for off‐line analysis.Monitor resting membrane potential and the action potential for more than 5 minutes before drug applications.
?. [Voltage-Clamp Recording]
Visualize their neuronal somata through a ×40 water immersion objective using IR differential interference contrast optics.
?. [Voltage-Clamp Recording]
Add Tetraethylammonium (TEA) and ZD 7288 (Sigma‐Aldrich) the artificial cerebrospinal fluid, to block K+ orIhcurrents, respectively.
TITLE: Light microscopy immunoperoxidase staining protocol
AUTHORS: Yoland Smith

[DESCRIPTION]
This protocol details the procedure of light microscopy immunoperoxidase staining protocol.

[STEPS]
SECTION: Light microscopy immunoperoxidase staining protocol
1. Select sections to process from the brain tissue bank.
SECTION: Light microscopy immunoperoxidase staining protocol
2. Wash sections thoroughly with a phosphate-buffered saline(PBS, 0.01 Molarity (M), pH 7.4) solution.
SECTION: Light microscopy immunoperoxidase staining protocol
3. Treat the sections at Room temperature with a 1% sodium borohydride (NaBH4) solution in PBS for 20 min.
SECTION: Light microscopy immunoperoxidase staining protocol
4. Rinse sections thoroughly in PBS.
SECTION: Light microscopy immunoperoxidase staining protocol
5. Pre-incubate sections for 60 min at Room temperature in a solution containing 1% normal serum (from the species used to generate the secondary antibodies), 0.3% Triton X-100, and 1% bovine serum albumin (BSA) in PBS.
SECTION: Light microscopy immunoperoxidase staining protocol
6. Incubate the sections for 1440 min at Room temperature in a solution containing the primary antibodies in 1% normal serum, 0.3% Triton X-100, and 1% BSA in PBS.
SECTION: Light microscopy immunoperoxidase staining protocol
7. Next day, thoroughly rinse the sections in PBS.
SECTION: Light microscopy immunoperoxidase staining protocol
8. Incubate the sections in a PBS solution containing the appropriate biotinylated secondary antibody (1:200; Vector Labs, Burlingame, CA, USA) combined with 1% normal serum, 0.3% Triton X-100, and 1% BSA for 90 min at Room temperature.
SECTION: Light microscopy immunoperoxidase staining protocol
9. Wash the sections in PBS.
SECTION: Light microscopy immunoperoxidase staining protocol
10. Incubate the sections in an avidin-biotin-peroxidase complex (ABC; 1:100; Vector Labs, Burlingame, CA, USA) solution for 90 min at Room temperature.
SECTION: Light microscopy immunoperoxidase staining protocol
11. Rinse the sections in PBS twice followed by a third rinse in TRIS buffer (0.05 Molarity (M); pH 7.6).
SECTION: Light microscopy immunoperoxidase staining protocol
12. Incubate the sections in a solution containing 0.025% 3,3ʹ-diaminobenzidine tetrahydrochloride, 10 millimolar (mM) imidazole, and 0.005% hydrogen peroxide in Tris buffer for 10 min at Room temperature.
SECTION: Light microscopy immunoperoxidase staining protocol
13. Rinse the sections with PBS.
SECTION: Light microscopy immunoperoxidase staining protocol
14. Mount sections onto gelatin-coated slides, and coverslip with Permount.
SECTION: Light microscopy immunoperoxidase staining protocol
15. Digitalize the slides with an Aperio ScanScope CS system (Aperio Technologies).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The NEBNext Ultra II DNA Library Prep Kit for Illumina contains the enzymes and buffers required to convert a broad range of input amounts of DNA into high quality libraries for next-generation sequencing on the Illumina platform. The fast, user-friendly workflow also has minimal hands-on time.</p>

[BEFORE_START]
<p><strong>Starting Material:</strong> 500 pg–1 μg fragmented DNA. We recommend that DNA be sheared in 1X TE. If the DNA volume post shearing is less than 50 μl, add 1X TE to a final volume of 50 μl. Alternatively, samples can be diluted with 10 mM Tris-HCl, pH 8.0 or 0.1X TE.</p>

[STEPS]
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TITLE: Differentiation of mature neurons from mouse NPCs
AUTHORS: Sonia Cruciani, Jacqueline Severo, Laura Batlle, Eva Maria Novoa

[DESCRIPTION]
This is a step by step protocol used to differentiate  mouse NPCs to mature neurons. 

[STEPS]
SECTION: Coating
1. -Dilute Poly-L-Ornithine (PLO) 1:6 in H2O
SECTION: Coating
2. -Fully coat the wells with diluted PLO (i.e. 500 ul per well in a 12WP)
SECTION: Coating
3. -Leave in the incubator for minimum 2h (also overnight works)
SECTION: Coating
4. -Aspirate the PLO and wash three times with sterile H2O
SECTION: Coating
5. -Dilute Laminin 1:500 in sterile H2O
SECTION: Coating
6. -Fully coat the wells with diluted Laminin (i.e. 500 ul per well in a 12WP)
SECTION: Coating
7. -Leave in the incubator for minimum 2h (also overnight works)
SECTION: Plating - Day -1
8. -count the desired number of cells:  Plate ~100.000 cells on a coverslip, ~150.000 cells in a 24WP well, ~250.000 cells in a 12WP well.
SECTION: Plating - Day -1
9. -seed NPCs for the final differentiation in NPC medium
SECTION: Day 0
10. -aspirate all NPC medium
SECTION: Day 0
11. -immediately add differentiation medium not directly on the bottom of the well but making it go through the walls to avoid disturbing the cells.
SECTION: Day 2-14
12. -Change medium (remove half volume and add half volume of new medium) avoiding the cells’ contact with air every 2-3 days. If at diff day 2-3 you notice overgrowth of glial cells, add AraC 1uM to the media in order to stop proliferation.
Cells can be kept in colture for >14 days, but they are already MAP2+ and TUJ1+ at day 7.
SECTION: Tamoxifen treatment
13. -Add 2.5 uM Tamoxifen to the medium, and the corresponding volume of metOH as control. Incubate for at least 6 days.
TITLE: 3D Mesh Cleanup Tutorial
AUTHORS: Elizabeth G. Clark, Kelsey M Jenkins, Craig R Brodersen

[DESCRIPTION]
This is the collection of protocols for developing 3D reconstructions of animals and plants from digitized specimens (see Clark et al. 2023). 3D Object files for use in the protocols are available at https://doi.org/10.6084/m9.figshare.21266568

[GUIDELINES]
This is the associated tutorials for Back to life: Techniques for developing high-quality 3D reconstructions of plants and animals from digitized specimens

This includes:

Beginner tutorial: Fossil plant cupule (Petriellaea triangulata [Shi et al. 2021])

Included files: Cupule Cleanup Tutorial.docx
3D object files: Cupule before.stl, Cupule before.obj
Necessary software: Meshlab
Skills developed: Shrinkwrapping, simplification

Intermediate tutorial: Reptile (Sphenodon punctatus [downloaded from Morphosource.org])

Included files: Sphenodon Cleanup Tutorial.docx 
3D object files: Sphenodon with holes.stl, Sphenodon with holes.obj
Necessary software: Meshlab, Autodesk Maya, Autodesk Fusion 360
Skills developed: Rearticulation, repair of broken fossils, basic sculpting, shrinkwrapping.

Advanced tutorial: Fossil crab (Cretapsara athanata[Luque et al. 2021)]

Included files: Crab Cleanup Tutorial.docx
3D object files: Crab before.stl, Crab before.obj
Necessary software: Meshlab, Autodesk Maya
Skills developed: Debris removal, structural repair, shrinkwrapping, rearticulation, smoothing and simplification

Before starting: 

Download 3D object files and install Meshlab, Autodesk Maya, and Fusion 360. 

Download 3D Object files at https://doi.org/10.6084/m9.figshare.21266568

Meshlab is an opensource software available at https://www.meshlab.net/

Autodesk Maya and Fusion 360 are available with a student and educators license at https://www.autodesk.com/education/edu-software/overview

[STEPS]
TITLE: Saliva Vapor Tests in a Home Environment for Point-of-care COVID-19 Tests
AUTHORS:  Shadi Emam, nasrollahpourmotla.m , n.sun 

[STEPS]
?. Saliva collection: the person to be tested by the COVID-19 sensor uses a one-time pipette for collecting a couple of drops (0.1mL) of saliva.
?. The pipette with a couple of drops of saliva will be placed at 1-inch distance beneath the SARS-COV-2 gas sensor in the handheld system with its sensor cover open.
?. Wait for 1~60 seconds, the handheld COVID-19 sensor system will show if the test result is shown in the LED indicator with a red (positive), green (negative), or yellow (undecided) light on. The person who is tested with a yellow on will be recommended to a local COVID-19 test site for a molecular test.
?. All test data will be automatically sent to the smartphone that is connected to the handheld system for data collection and routing to NIH RADx DCC automatically.
?. Turn on the handheld COVID-19 test system. Open the COVID-19 Sensor Mobile App on the smartphone and connect the handheld sensor to the smartphone via Bluetooth.
?. Disposal of the pipette with saliva after rinsing it with Lysol thoroughly for 10 minutes.
TITLE: qPCR and RT-PCR
AUTHORS: Ayse Ulusoy, Michael Klinkenberg, Michael Helwig, Angela Rollar, Shirley Lee, Rita Pinto-Costa, Donato Di Monte

[DESCRIPTION]
The protocol describes the methodology and materials we use in the Di Monte lab for mRNA extraction and q-PCR/or RT-PCR analyses from fixed tissue samples. 

[STEPS]
1. Dissect the dorsal medulla oblongata from paraformaldehyde-fixed coronal sections
of the medulla oblongata.


2. Extract total RNA using Nucleic Acid Isolation Kit (Ambion), and measure yield using a nanodrop.

2.3. Mix the PCR products with 6x sample buffer (New England
Biolabs) and 5% DMSO and load on 2.0% SeaKem agarose gel
(Lonza Bioscience) pre-stained with RedSafe dye (1:20,000, Intron
Biotechnology)
2.2. Run a 30-cycle PCR reaction. 
SECTION: Conventional reverse trancription polymerase chain reaction (RT-PCR)
2.1. Synthesize cDNA from 100 ng template RNA using SuperScriptVILO Master Mix (Life Technologies) and the suitable primers (20 µLfinal volume).

2.6. Run the PCR with a real-time PCR machine (e.g., StepOnePlusTM real-time PCR system, Applied
Biosystems) in triplicates. 
2.5. Prepare a 20 µLl reaction mix containing 1 ml
cDNA, Power SYBRGreen (Applied Biosystems) and
primers. 
SECTION: real-time PCR
2.4. If necessary, extract RNA from human brain (Agilent Technologies) as a
reference sample.
TITLE: U Michigan - Retinal Cell Death
AUTHORS: David A. Antonetti

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Capture nucleosomes from the cytoplasm of apoptotic cells on a micro titer plate and detect the associated histones with HRP-conjugated antibody. Designed for use with cell culture but adapted for use with homogenized mouse retinas.</div></div>

[STEPS]
?. Weigh the microcentrifuge tube
?. Add the retina sample and weigh the retina with tube to obtain the retinal weight
?. Freeze the retina in liquid nitrogen and store at -80°C (or use fresh retina)
?. Add 50 µl 1x Lysis buffer to each mouse retina
?. Homogenize the retina and keep it on ice
?. Vortex and incubate the sample at room temperature for 30 minutes with gentle rocking
?. Centrifuge at 12500 rpm at 4ºC for 10 minutes
?. Collect the supernatant in fresh tube and save the pellet
?. Add 20 µl of samples, positive control (supplied in kit) and blank or negative control (lysis buffer) in each well (triplicates)
?. Add 80 µl of Immuno reagent to each well
?. Cover the plate with adhesive strip, cover with foil and incubate with gentle shacking (300 rpm) at room temperature for 2 hours
?. Carefully remove the solution by aspiration
?. Aspirate each well and wash by filling each well with 200 µl of incubation buffer 1x (bottle #4) using multi-channel pipette
?. Repeating the washing process for a total of 5 washes
?. Add 100 µl of ABTS Solution to each well and incubate on plate shaker (protect from light) until color develops (about 10 min)
?. Add 100 µl of ABTS stop solution to each well and gently tap the plate to ensure thorough mixing
?. Read the plate within 30 minutes at 405 nm using ABTS solution plus 100 µl of ATBS stop solution as blank (use reference wavelength 490 nm)
TITLE: Protocol for Nuclei/ Cell Isolation and 10X Genomics Fixed RNA profiling for Human Ovary
AUTHORS: Nicolas Martin

[DESCRIPTION]
This is the 10X Genomics protocol to fix, dissociate, and profile RNA from human ovary tissue. 

[GUIDELINES]
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).

[STEPS]
SECTION: Cell/Nuclei Isolation Protocol for Human Ovary
1. The protocol CG000553 REV B was used to fix, dissociate, and isolate cells/nuclei from frozen human skeletal muscle with the following modifications: 

1) 0.25 mg / mL of Liberase TH was used for dissociation at Step 2b, Page 6.
2) Two extra "spin only" (i.e., steps 3 and 4 ) of the Octodissociator protocol were run for each sample due to the presence of intact/ large tissue pieces at the end of the run at Step 2c, Page 6.   
3) Counts were performed using the Cellaca PLX Automated Cell counter (PN: PLX-SYS1) and ViaStain AOPI staining solution (PN: CS2-0106-5mL) at Step 2i on page 6. 

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/sample-prep/tissue-fixation-and-dissociation-for-chromium-single-cell-gene-expression-flex


SECTION: Chromium Fixed RNA Profiling Reagent Kits
2. The protocol CG000527 Rev E  was used to generate gene expression libraries from fixed cell/nuclei suspension inputs.

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/library-prep/chromium-single-cell-gene-expression-flex-reagent-kits-for-multiplexed-samples

Samples were multiplexed in batches of 16 using the user guide CG000565. 

https://www.10xgenomics.com/support/single-cell-gene-expression-flex/documentation/steps/library-prep/chromium-single-cell-gene-expression-flex-reagent-kits-for-multiplexed-samples
TITLE: UC Davis - Leptin Signaling pathway
AUTHORS: Fawaz G. Haj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
This test is designated to determine if rodents exhibit activation of leptin signaling pathway through evaluation of the phosphorylation state of JAK2 and STAT3 proteins. </div></div>

[STEPS]
?. Unless otherwise requested by the PI or stated in the protocol, mice will be euthanized using cervical dislocation.
?. Collect maximum blood from portal vein and isolate plasma according to standard protocols or as desired by the P.I.
?. Quickly collect tissues designated by the P.I. Each tissue should be divided into three portions, one portion should be snap frozen in liquid nitrogen, one portion should be kept into RNA later solution and the third one should be fixed into the appropriate fixative solution. Please note that the whole procedure of tissue collection should be done within 3 minutes maximum.
?. For western blotting, tissues will be lysed into the appropriate lysis buffer.
?. Total protein expression of Leptin Receptor, JAK2, and STAT3, phosphorylation of JAK2 and STAT3 in tissues of interest will be determined according to the standard Western blotting protocols.
?. Note:
Evaluation of the activation state of other component of the leptin signaling and other associated signaling pathways is also possible upon special request. Extra charges may apply.
Gene expression of proteins involved in leptin signaling is feasible if requested by the P.I. Extra charges may apply.
Immunohistochemistry of leptin signaling could be performed on fixed tissues if desired by the P.I. Extra charges may apply.
TITLE: NaOH裂解基因组
AUTHORS: 张 雪

[STEPS]
?. 拔鱼鳞＋50µl 50mM NaOH，95°C，15min
95 °C
?. 吹散混匀后，+5µl pH8.0  1M Tris-HCl，此为stock（-30°C储存）
?. 取10µl stock+20µl ddH2O，此为work