eamag/protocols-with-steps huggingface dataset:
TITLE: Populating NCBI template for submissions using  BioNumerics v7.6
AUTHORS: Ruth Timme, Maria Balkey, Julie Haendiges

[DESCRIPTION]
PURPOSE: to define the standard operating procedure for collecting isolate metadata using BioNumerics for submission of food/environmental isolates to NCBI.

SCOPE: to provide a standardized procedure to collect isolate metadata using BioNumerics for submission of food/environmental isolates to NCBI.

RESPONSIBILITIES- SOP Responsible Officials: Ruth Timme, Maria Balkey

The GenomeTrakr Network Management will be responsible to monitor GenomeTrakr submissions processed through Bionumerics and ensure that all GT labs are familiar with the mandatory metadata fields required for submission of GenomeTrakr sequencing records to NCBI. 

V3:  Dropdown menus from controlled vocabulary added to the ncbi_update submission sheet

[STEPS]
SECTION: New Version for this protocol
1. Visit the new version of this protocol by clicking the following link: https://www.protocols.io/view/populating-ncbi-template-for-submissions-using-bio-3byl4qn4ovo5/v1
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Protocol for transforming XL-10 Gold Ultracompetent cells from Stratagene (now Agilent). Protocol adopted from manufacturer's instructions and simplified to remove use of special media and beta-mercaptoethanol

[GUIDELINES]
<strong>Use of 14-ml BD Falcon polypropylene round-bottom tubes</strong>: It is important that 14-ml BD Falcon polypropylene round-bottom tubes (BD Biosciences Catalog #352059) are used for the transformation protocol, since other tubes may be degraded by β²-mercaptoethanol. In addition, the duration of the heat pulse has been optimized using these tubes.<br /><strong>Aliquoting Cells</strong>: Keep the cells on ice at all times during aliquoting. It is essential that the polypropylene tubes are placed on ice before the cells are thawed and that the cells are aliquoted directly into pre-chilled tubes. It is also important to use 100 μl of cells per transformation. Decreasing the volume will reduce efficiency.<br /><strong>Use of Î²-Mercaptoethanol (β²-ME)</strong>: β²-ME has been shown to increase transformation efficiency. The β²-ME mixture provided is diluted and ready to use. Stratagene cannot guarantee results with Î²-ME from other sources.<br /><strong>Use of NZY+ Broth</strong>: Transformation of the supplied ultracompetent cells has been optimized using NZY+ as the medium for outgrowth following the heat pulse. Substitution with another outgrowth medium may result in a loss of efficiency.<br /><strong>Quantity and Volume of DNA</strong>: The greatest efficiency is obtained from the transformation of 1 μl of 0.01 ng/μl supercoiled pUC18 DNA per 100 μl of cells. When transforming a ligation mixture, add 2 μl of the ligation mixtureper 100 μl of cells. A greater number of colonies may be obtained by transforming up to 50 ng DNA, although the resulting efficiency (cfu/μg) may be lower. The volume of the DNA solution added to the reaction may be increased to up to 10% of the reaction volume, but the transformation efficiency may be reduced.<br /><strong>Heat Pulse Duration and Temperature</strong>: Optimal transformation efficiency is observed when cells are heat-pulsed at 42°C for 30 seconds. Efficiency decreases sharply when cells are heat-pulsed for &lt;30 seconds or for &gt;40 seconds. Do not exceed 42°C.<br /><strong>Plating the Transformation Mixture</strong>: If plating &lt;100 μl of cells, pipet the cells into a 200 μl pool of medium and then spread the mixture with a sterile spreader. If plating ≥100 μl, the cells can be spread on the plates directly. Tilt and tap the spreader to remove the last drop of cells. If desired, cells may be concentrated prior to plating by centrifugation at1000 rpm for 10 minutes followed by resuspension in 200 μl of NZY+ medium or alternative medium.

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TITLE: Intracellular neuromelanin quantification
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Protocol for quantifying intracellular neuromelanin in coronal sections of the rodent brain, in HE stained sections (NM-occupied area) and unstained sections (NM OD).

[STEPS]
SECTION: H&amp;E-stained sections (NM-occupied neuronal area)
1. Scan H&E sections using the Pannoramic Midi II FL, HQ SCIENTIFIC 60x and section images were acquired with CaseViewer software at an objective magnification of 63x. 
SECTION: H&amp;E-stained sections (NM-occupied neuronal area)
2. Acquire SNpc images at 63x with CaseViewer.
SECTION: H&amp;E-stained sections (NM-occupied neuronal area)
3. Upload individual images at Image J software.
SECTION: H&amp;E-stained sections (NM-occupied neuronal area)
4. Click on Adjust canvas size and adjust it at 1596x1198.
SECTION: H&amp;E-stained sections (NM-occupied neuronal area)
5. Click on Invert image.
SECTION: H&amp;E-stained sections (NM-occupied neuronal area)
6. With the free hand selections tool, draw a neuromelanin-pigmented neuron cytoplasm (excluding the nucleus) and measure the area.
SECTION: H&amp;E-stained sections (NM-occupied neuronal area)
7. With the free hand selections tool, draw the neuromelanin pigment of the neuron and measure the area.
SECTION: H&amp;E-stained sections (NM-occupied neuronal area)
8. Calculate the percentage of the neuronal area occupied by the pigment using the formula: neuromelanin pigment area/neuronal cytoplasm area.

SECTION: Unstained sections (NM optical density)
13. Calculate the optical density of the neuromelanin pigment for each animal using the mean values of the pigmented neurons in the unstained sections.
SECTION: Unstained sections (NM optical density)
9. For unstained sections, take pictures of different fields in the pigmented area using the Zeiss Imager.D1microscope coupled to an AxioCamMRc camera. 
SECTION: Unstained sections (NM optical density)
10. Upload individual images at Image J software.
SECTION: Unstained sections (NM optical density)
11. Click on Invert image.
SECTION: Unstained sections (NM optical density)
12. With the free hand selections tool, draw the intraneuronal neuromelanin pigment of a neuron and measure the optical density. Measure approximately 30-50 neurons per animal, depending on the brain region.
TITLE: Patch-Seq Recording and Extraction Detailed Protocol
AUTHORS: Brian Lee, Kristen Hadley, Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is a detailed version of the Allen Institute's Patch-Seq Recording and Extraction protocol, which describes the process to obtain electrophysiological recordings and cellular contents from neurons in postnatal mouse and/or human brain slices.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Seminavis robusta protocol collection
AUTHORS: Lev Tsypin, Aaron Turkewitz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a collection of media, reagents, and protocols for the cultivation and manipulation of the diatom </span><span style = "font-style:italic;">Seminavis robusta</span><span>. Our hope is that these documents will help future researchers develop genetic tools for studying this organism.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>GreenGlo ™, 20,000X in Water, is a non-carcinogenic and non-toxic alternative to Ethidium bromide used for the detection of nucleic acids in agarose gels. It is as sensitive as Ethidium bromide. There is no toxic DMSO as GreenGlo ™ is supplied in water. <br /> <br />GreenGlo ™ has fluorescence excitation maxima at 295 nm and 490 nm. The fluorescence emission maxima is similar to EtBr when bound to DNA – at 530 nm.</p>

[STEPS]
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TITLE: Supplemented M9 media
AUTHORS: Wolfram Moebius

[DESCRIPTION]
Supplemented M9 media

[STEPS]
1. 
For 500 mL need:

Dissolve 56.4 g M9 salts (5X) in MilliQ water, make up to 1L and autoclave
Dissolve 10 g of casamino acids in MilliQ water, make up to 10 mL and filter with a    filter 
5 mL 40% w/v glucose, autoclaved
1 mL 1M MgSO4, filter sterilised
50 uL 1M CaCl2, filter sterilised
50 uL 1% w/v thiamine hydrochloride, filter sterilised
RO water up to 500mL, autoclaved

Add ingredients to a sterile 500 mL bottle, if any salts precipitate heat to 80 °C and swirl until dissolved.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the suggested protocol for use with &lambda; DNA-Mono Cut Mix (<a href="https://www.neb.com/products/n3019-dna-mono-cut-mix" target="_blank">N3019</a>),&nbsp;фX174 DNA-HaeIII Digest (<a href="https://www.neb.com/products/n3026-x174-dna-haeiii-digest" target="_blank">N3026</a>),pBR322 DNA-BstNI Digest (<a href="https://www.neb.com/products/n3031-pbr322-dna-bstni-digest" target="_blank">N3031</a>), pBR322 DNA-MspI Digest (<a href="https://www.neb.com/products/n3032-pbr322-dna-mspi-digest" target="_blank">N3032</a>), 2-Log DNA Ladder (0.1-10.0 kb) (<a href="https://www.neb.com/products/n3200-2-log-dna-ladder-01-100-kb" target="_blank">N3200</a>),&nbsp;100 bp DNA Ladder (<a href="https://www.neb.com/products/n3231-100-bp-dna-ladder" target="_blank">N3231</a>), 1 kb DNA Ladder (<a href="https://www.neb.com/products/n3232-1-kb-dna-ladder" target="_blank">N3232</a>), Low Molecular Weight DNA Ladder (<a href="https://www.neb.com/products/n3233-low-molecular-weight-dna-ladder" target="_blank">N3233</a>), and 50 bp DNA Ladder (<a href="https://www.neb.com/products/n3236-50-bp-dna-ladder" target="_blank">N3236</a>)

[BEFORE_START]
The following protocol is recommended for a 5 mm wide lane.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Invitrogen's <a href="https://www.thermofisher.com/antibody/product/HA-Tag-Antibody-clone-2-2-2-14-Monoclonal/26183" target="_blank">HA tag monoclonal antibody</a> in combination with <a href="https://www.thermofisher.com/order/catalog/product/WB7104" target="_blank">Western Breeze Anti-Mouse Chemiluminescent Western Blot Immunodetection Kit</a> leads to no background Western blots with Pt lysates. HA tag thus represents a tag of choice for (1) efficient and unambioguous protein expression analysis, (2) immunofluorescence experiments and (3) various IP and pull-down experiments in Pt and possibly other diatoms as well as other marine microeukaryotes.      </p>

[BEFORE_START]
<p>The starting point of this protocol is a genotype+ genetically engineered <em>Pt</em> strain. </p>

[GUIDELINES]
<p>Recommended HA tag nucleotide sequence is as follows: 5'-TATCCGTATGATGTCCCCGATTACGCG-3'.  </p>

[STEPS]
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TITLE: General Guidelines for Culture of Multiple Myeloma Cell Lines
AUTHORS: Dennis Juarez, dfruman

[DESCRIPTION]
General guidelines for culture and use of myeloma cell lines in the Fruman Lab.

[STEPS]
1. Incubator Settings: 37 degrees Celsius, 5% CO2
2. Medium: 88% RPMI 1640, 10% FBS, 1% Pen/Strep/Glutamine, 1% (Final: 10 mM) of 1M HEPES
3. Cryopreservation: Freeze medium: 90% FBS, 10% DMSO. 5 million cells are resuspended in 500uL of freeze media, transferred to cryotubes, and frozen slowly in Mr. Frosty™ Freezing Container at -80 degrees Celsius.
4. Guidelines for Experimental Use:
Passages are not intuitive for suspension cells and passage number should instead be used to indicate number of times the original stock has been grown out and frozen for future experiments.
New cell lines should be grown to at least 35 million cells and a minimum of 6 vials should be frozen for experimental use throughout the year and keeping at least one for long term storage in liquid nitrogen. 
Cell lines should be authenticated through STR profiling annually or whenever a new set of stocks are cryopreserved for future experiments to confirm lack of cross contamination.
Cell lines should not be cultured for longer than 2 months when used for experiments.
Cell lines should be maintained at concentrations between 0.5 million to 2 million per mL throughout culture as to not exhaust media.
Experiments requiring incubation times less than 24 hours should have cells plated at 1 million per mL media.
Experiments requiring incubation times greater than 24 hours should have cells plated at 0.25-0.5 million per mL media.
When ordering new lots of FBS, a sample should be requested to test if the new lot will be deleterious to cell survival and cell proliferation.
TITLE: Untargeted Top-down Proteomics by LC-MS/MS on Eclipse
AUTHORS: Bryon  Drown, Jeannie  Camarillo, Rafael Melani, Neil  Kelleher

[DESCRIPTION]
Describes the LC-MS/MS data acquisition procedure for top-down proteomics samples using the Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer

[STEPS]
1. Samples were analyzed on a Thermo Scientific Orbitrap Eclipse Tribrid mass spectrometer in line with a Dionex Ultimate 3000 RSLCnano system
2. Samples (6 µL ) were injected via the autosampler and loaded onto a self-packed trap column (150 μm i.d. x 2 cm length packed with PLRP-S 5-μm particles 1,000-Å pore size) for 10 min with 100% loading buffer (94.8% water:5% acetonitrile:0.2% formic acid)  at 3 uL/min.

3. Following a valve switch and initiation of the nanopump at 300 nL/min (buffer A: 94.8 % water, 5 % acetonitrile, 0.2 % formic acid; buffer B: 4.8 % water, 95 % acetonitrile, 0.2 % formic acid), proteins were separated on a self-packed analytical column (75 μm i.d. x 25 cm length packed with PLRP-S 5-μm particles 1,000-Å pore size) according to the following gradient for fractions 1-4:
 
 %BValve position  0  51_1010  51_2121562556795729575  590  5
 
For fraction 5 and later, nanopump used the following gradient:

 

 
4. Eluted proteins were ionized in positive ion mode nanoelectrospray ionization (nESI) using a pulled tip nanospray emitter (15-μm i.d. ×125 mm) packed with 1mm of PLRP-S 5-μm particles 1,000-Å pore size with a custom nano-source (https://proteomicsresource.washington.edu/docs/protocols05/UWPR_NSI_Source.pdf).


 

5. Precursor (intact protein) spectra were acquired at 120k FTRP.

 

6. The mass spectrometer was operated using a TopN 3 sec data-dependent acquisition mode
Precursor ions were filtered by intensity, charge state, and dynamic exclusion:

 

7. Ions for fragmentation were isolated and fragmented via higher energy dissociation (HCD):
TITLE: Microplate-based DNA Quantification with EzFluoroStain DNA reagent
AUTHORS: Maho Saita, Kyoko Aikawa, Kenji  Ohgane

[DESCRIPTION]
This protocol offers an safer alternative to the ethidium bromide-based DNA quantification protocol, utilizing an DNA-selective dye EzFluoroStain DNA (WSE-7130, ATTO corporation, Tokyo, Japan). This protocol allows to quantify about 2-1000 ng DNA/well, using a standard fluorescent plate reader. 

[BEFORE_START]
We recommend to aliquot EzFluoroStain DNA reagent in ~10µL and minimize freeze-thaw cycles (several freeze-thaw cycles are OK). Before starting the experiment, thaw an aliquot of EzFluoroStain DNA reagent while protecting from light.

[GUIDELINES]
    This DNA quantification protocol is based on interaction of a fluorogenic dye EzFluoroStain DNA (ATTO Corporation, Japan) with DNA. The exact structure of this commercial dye is not disclosed, the mechanism is similar to intercalating dyes ethidium bromide (Bonasera et al., 2007), SYBR Green (Leggate et al., 2006), and PicoGreen (Singer et al., 1997). Although some commercial reagents for DNA quantification, including PicoGreen, offer higher sensitivity, this dye can be a cheaper alternative sufficient for routine use. In our laboratory, we routinely use EzFluoroStain DNA for both DNA quantification and agarose gel staining.
    Compared with UV-based measurement, this protocol allows determination of lower concentration of DNA, with relatively small amount of the DNA solution (1-10 µL).
    This protocol allows the users to quantify about 2-1000 ng DNA per well. However, the dynamic range may vary depending on different plate readers and settings (especially for lower concentration range). The detection settings/methods may need to be optimized for this assay, using standard samples.
    In the protocol, 5 µL sample / well was used as a standard condition. However, the amount of the sample can be varied depending on the estimated concentration of the samples. For example, 0.4 to 200 ng/µL DNA solution can be measured with this 5 µL/well protocol, and 2 to 1000 ng/µL DNA solution can be assayed with 1 µL/well protocol. Note that more than 1000 ng DNA per well results in saturation of the fluorescence, as shown in the example data below.

 

 
References
 

 

 


[STEPS]
SECTION: Sample preparation
1. Prepare dilution series of standard DNA solution, depending on the concentration of your sample.

 

SECTION: Sample preparation
2. Prepare EzFluoroStain DNA working solution.

 

SECTION: Sample preparation
3. Mix 5 µL of the DNA samples with 95 µL of the EzFluoroStain DNA working solution on a black 96-well microplate.

 


SECTION: Measurement &amp; analysis
4. Measure fluorescence.

 

SECTION: Measurement &amp; analysis
5. Calculate the concentration of your samples using standard curve.
TITLE: High-Capacity cDNA Reverse Transcription
AUTHORS: Hector Martell Martinez

[DESCRIPTION]
This protocol details the high-capacity cDNA reverse transcription.

[STEPS]
SECTION: Nanodrop
1. Nanodrop each isolated sample of RNA.
SECTION: Nanodrop
1.1. A good concentration of RNA is between 200 3172- 2000 3172 .

If the concentration is above 2000 3172 then dilute the sample with water for a final concentration below2000 3172 .

SECTION: Nanodrop
1.2. A good 260/280 value is ~2.0.
SECTION: Nanodrop
1.3. A good 260/230 value is ~2.0-2.2.
SECTION: cDNA Calculations
2. cDNA for all brain regions is made with 2000 ng of RNA. cDNA for isolated cells is made with the highest amount of RNA that can be made from the least concentrated sample. 

TV per reaction = 20 µL (10 µL of RNA/Water + 10 µL of Master Mix)

Calculate the RNA amount needed to make 2000 ng of RN for each sample

Ex. For a RNA concentration of 1250.0 3172 .
2000/1250 = 1.6 µL of RNA

SECTION: cDNA Calculations
3. Calculate the amount of water to be added to the RNA for a TV = 10 µL

Ex. For 1.6 µL of RNA
10 µL – 1.6 µL of RNA = 8.4 µL of Water


 

SECTION: Making cDNA
4. Thaw the isolated RNA and the following components of the High-Capacity cDNA reverse transcription kit on ice.

10X RT Buffer
25X dNTP Mix (100 millimolar (mM) )
10X Random Primers – Can thaw at Room temperature
RNase Inhibitor


 

SECTION: Making cDNA
5. While the above components thaw on ice pipette the calculated amount of water (from   of cDNA calculations) to PCR tubes. This step can be done at  Room temperature.

SECTION: Making cDNA
6. Place the PCR tubes with water on ice and then add the calculated amount of RNA (from   of cDNA calculations) to its respective PCR tube.

SECTION: Making cDNA
7. Create the following master mix on ice.  Make enough master mix for each sample plus a little extra (if you have 10 samples, make enough master mix for 11).

SECTION: Making cDNA
7.1. Add reagents to a 1.5 mL tube in the following order.

 

SECTION: Making cDNA
7.2. Mix gently.
SECTION: Making cDNA
8. Add 10 µL of master mix to each PCR tube on ice.

SECTION: Making cDNA
9. Mix PCR tubes gently then spin down briefly.
SECTION: Making cDNA
10. Keep  on ice until performing the reverse transcription.
SECTION: Perform Reverse Transcription
11. Place PCR tubes into the thermal cycler.
SECTION: Perform Reverse Transcription
12. Set the Reaction Volume to 20 µL.

SECTION: Perform Reverse Transcription
13. Set the following conditions: 

 

SECTION: Perform Reverse Transcription
14. Start the thermal cycler run.
SECTION: Perform Reverse Transcription
15. When the samples reach   take the PCR tubes out and store at 4 °C for short term use and at -20 °C for long term use.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; background-color: #f6f6f4;">This tutorial demonstrates the use of VIROME for comparing viral metagenome libraries analyzed with the VIROME pipeline as well as comparative analysis of viral communities using the Quantitative Insights in Microbial Ecology package (QIIME). <br /><br />Wommack, K. E., J. Bhavsar, S. W. Polson, J. Chen, M. Dumas, S. Srinivasiah, M. Furman, S. Jamindar, and D. J. Nasko. 2012. VIROME: a standard operating procedure for analysis of viral metagenome sequences. Standards in Genomic Sciences 6:427-439 [</span><a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3558967/" target="_blank">PMC3558967</a><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; background-color: #f6f6f4;">] </span><br style="box-sizing: border-box; color: #000339; font-family: Raleway, sans-serif; font-size: 14px; background-color: #f6f6f4;" /><br style="box-sizing: border-box; color: #000339; font-family: Raleway, sans-serif; font-size: 14px; background-color: #f6f6f4;" /><a href="http://virome.dbi.udel.edu/" target="_blank">See site</a><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; background-color: #f6f6f4;"> for more information.</span>

[GUIDELINES]
<span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; background-color: #f6f6f4;">See full video below for more information. <br /><br /><iframe src="https://www.youtube.com/embed/mL0qRR1uYUM" width="420" height="315" frameborder="0" allowfullscreen="allowfullscreen"></iframe><br /><br /></span>Youtube link: <a href="https://www.youtube.com/watch?v=mL0qRR1uYUM" target="_blank">https://www.youtube.com/watch?v=mL0qRR1uYUM</a>

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