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

[DESCRIPTION]
Formulation for a phosphate-buffered 20% glutaraldehyde stock solution.

[GUIDELINES]
<table>
<tbody>
<tr>
<td>Solution A</td>
<td>Solution B</td>
</tr>
<tr>
<td>Na<sub>2</sub>HPO<sub>4</sub> 1.414g in 50ml 0.02µm filtered mQ</td>
<td>NaH<sub>2</sub>PO<sub>4</sub> 1.058g in 50ml 0.02µm filtered mQ</td>
</tr>
</tbody>
</table>

[STEPS]
?. 
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TITLE: gRNA POOL NGS SEQUENCING LIBRARY PREPARATION
AUTHORS: Renuka Ravi Gupta, Helaine Graziele Santos Vieira, Helen E. King, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Robert J Weatheritt, Lorenz Studer, Joseph Powell

[DESCRIPTION]
Quantification of the frequency of each sgRNA in the library pool is necessary before proceeding to the 10x Genomic assay. We prepare genomic DNA collected after lentiviral transduction of the CRISPR library in H9 dCAS9 cell line to verify the plasmid/viral pool for the CRISPR library via Next Generation Sequencing (NGS sequencing). Genomic DNA was extracted from cells of interest, PCR amplified and the final product was sent to the sequencing facility, where the downstream cleanup and sequencing was performed.

[BEFORE_START]
H9 dCAS9 CRISPRi were transduced with the pooled CRISPRi library to  obtain the gDNA (genomic DNA) after lentiviral transduction.
 


[STEPS]
SECTION: DNA Extraction
1. Collect cells after FACS sort in E8 flex media. 
SECTION: DNA Extraction
2. Centrifuge the cells at 300 g for 4 mins. 
SECTION: DNA Extraction
3. Aspirate the supernatant and freeze down the cell pellet or continue with DNA extraction using the QIAamp DNA Blood Mini Kit.
SECTION: DNA Extraction
4. Measure the concentration of the DNA using nanodrop to check the  quality of the DNA and using Qubit to get the precise concentration of the DNA
SECTION: PCR amplification
5. PCR reactions are set up as follows:
Template 50 ng pCRISPRi_dual guide gRNA library, 1:1 primers mU6 Fwd + RW704 illuSeq-Rev
Template 50 ng pCRISPRi_dual guide gRNA library, 1:1 primers LKO/U6 Fwd + RW705 illuSeq-Rev
SECTION: PCR amplification
6. Inorder to ensure enough yield of DNA product we load 50 ng of DNA/ reaction for the PCR amplification for each run and set up at least 4 PCR reactions which will be pooled to ensure even distribution of the sgRNAs.
  

 
Detailed protocol for PCR sample preparation can be found in this link: https://rochesequencingstore.com/wp-content/uploads/2023/02/KAPA-HiFi-HotStart-ReadyMix-PCR-Kit-Technical-Data-Sheet_v14-22.pdf

SECTION: PCR amplification
7. Set up the thermal cycler using the following program: 
95ºC for 3 minutes
25 cycles of: 
95ºC for 30 seconds
95ºC for 30 seconds
95ºC for 30 seconds
72ºC for 5 mins
Hold at 4ºC
Detailed protocol for PCR sample preparation can be found in this link: https://support.illumina.com/documents/documentation/chemistry_documentation/16s/16s-metagenomic-library-prep-guide-15044223-b.pdf

SECTION: PCR amplification
8. Pool the PCR products with similar forward and reverse primers together and send them to the sequencing platform.
SECTION: PCR amplification
9. QC for the final product was done by the sequencing platform by running the agarose gel to verify  the required size of band followed by downstream sequencing.
TITLE: Psychometric Properties of the Korean Version of the Health Literacy on Social Determinants of Health Questionnaire (K-HL-SDHQ)
AUTHORS: Mikyeong Cho, Hyeonkyeong Lee, Young-Me Lee, Ja-yin Lee, Haeyoung Min, Youlim Kim, Sookyung Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The association between the social determinant of health (SDH) and sustainable development goals, has directed attention toward the influence of SDH. However, there is a lack of evidence regarding the instruments used to assess SDH. Thus, this study was conducted to assess the validity and reliability of the Korean Version of the Health Literacy on Social Determinants of Health Questionnaire (K-HL-SDHQ). A total of 660 workers in Korea participated in an online survey. The K-HL-SDHQ measures four dimensions (Access, Understand, Appraise, and Apply) with 33 items. The HL-SDHQ was translated into Korean using the forward-back translation method. To test the validity and reliability of the Korean translated HL-SDHQ, item analysis for the 33 items was conducted. Internal consistency was examined using Cronbach’s ⍺, an exploratory factor analysis, and a confirmatory factor analysis. The scale-level content validity index (S-CVI)/Universal agreement of this study was .12 and S-CVI/Average variance extracted was .83 (item-CVI range=.50-1.00). The goodness of fit determined through a confirmatory factor analysis of the four dimensions was acceptable (χ</span><span style = "vertical-align:super;">2 </span><span>(489) = 1475.054, </span><span style = "font-style:italic;">p </span><span>< .001, RMSEA =.06, CFI =.87, TLI = .85). The K-HL-SDHQ also demonstrated satisfactory internal consistency reliability (Cronbach’s ⍺=.92). The findings indicate that the K-HL-SDHQ is a valid and reliable tool that can be used to assess the SDH of workers in Korea. It is suggested that this tool can be applied through repeated research with workers and non-workers for health promotion, and to enhance researchers’ understanding of the different levels of the HL-SDHQ. </span></div></div>

[STEPS]
TITLE: JAX - 10x Genomics - NEXTGEM v3.1 - 3&#39; Gene Expression
AUTHORS: William F F F. Flynn

[DESCRIPTION]
Standard 10x Genomics - NEXTGEM v3.1 - 3' protocol used by the JAX Single Cell Biology lab for single cell transcriptomics.

[STEPS]
1. Follow the 10x Genomics NEXTGEM v3.1 single cell 3' gene expression protocol.
TITLE: protocols.io for startups
AUTHORS: protocols.io team, protocols.io team

[DESCRIPTION]
We understand the financial constraints of young biotechs and other research-intensive startups. At the same time, it is important to establish early on the best practices around organizing and collaborating on development of research protocols that are central to the company's success.

In light of this, we offer our Startup Program that provides a full protocols.io Enterprise industry license at a deeply discounted annual price.

Please see below for the benefits, costs, eligibility, and application details.

[STEPS]
SECTION: Benefits
1. Unlimited private workspaces and protocols
Dedicated customer support
Customized training (2 training workshops per year and unlimited 1-on-1 demos)
Import of existing protocols
HIPAA support
Audit trail and electronic signature for 21 CFR Part 11

 

SECTION: Price
2. The annual Enterprise cost for startups is $5,000.


 

SECTION: How to apply
4. Simply send an email to info@protocols.io with:
Subject plan: Startup Program Application
Your name
Name of the company
Website address of the company

 

SECTION: Eligibility
3. The program is meant for companies with:
20 or fewer scientists
Less than $25M in venture capital funding
TITLE: Plant RNA extractions using TRI reagent
AUTHORS: Peter Crisp, Diep Ganguly, Aaron Smith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Extraction of total RNA from plant tissue using TRI reagent.</div></div>

[STEPS]
?. Collect 50-100 mg of plant tissue and freeze immediately in liquid N2.
?. Invert each tube by hand ~20x and incubate at room temperature for 5 minutes (DO NOT vortex samples as it may result in RNA degradation).
?. Centrifuge at 14,000 rcf for 10 minutes at 4°C.
?. Repeat steps 4 and 5 (approx. 400 µL).Note, if you are observing buffer and salt carryover in your purified RNA (high 230 nm absorbance), reduce volume of upper-phase recovered. Alternatively, replace chloroform (step 3) with chloroform : IAA (24:1).
?. Add equal volume ice-cold 100% isopropanol, then mix by inverting tubes ~20x by hand.Note, if you want to avoid capturing shorter RNA molecules (small RNAs, degradation intermediates), replace Isopropanol with 5 M LiCl (room temperature).
?. Centrifuge samples at 14,000 rcf for 20 minutes at 4°C.
?. Add 1 mL of 75 % ethanol and invert tube ~10x.
?. Remove supernatant (careful, pellet often becomes dislodged at this step).
?. Air-dry pellet at room temperature for 5 minutes.
?. If pellet is not dissolving straight away, incubate in buffer at room temperature for 5 minutes then resuspend by pipetting.
?. Visualise approx. 50 - 100 ng of purified RNA for each sample on a semi-denaturing 1% agarose TBE gel.
?. Grind tissue to fine powder under liquid N2 using tissue lyser or mortar + pestle, then immediately add 1 mL TRI reagent (1 mL per 100 mg tissue).Note, achieving a fine grind is critical to high yields of intact RNA.
?. Transfer the upper aqueous phase to a new microfuge tube (approx. 600 µL).
?. Incubate at -20°C for 1 hour. Alternatively, incubate O/N for small RNA precipitation or higher yields.
?. Add of chloroform (200 µL per mL TRI reagent), cap tubes, shake vigorously (by hand) at room temperature for 15 seconds (solution should become cloudy), then incubate at room temperature for 3 minutes.
?. Remove the supernatant.Note, you should observe a white pellet at this step, a yellow pellet is indicative of buffer carryover and you may need to do an additional clean-up.
?. Centrifuge samples at 7, 500 rcf for 5 minutes at room temperature.
?. Resuspend pellet in 25 - 100 µL of RNase-free (e.g. 0.01% DEPC-treated) buffer, depending on expected yield and desired concentration.Buffer = water or 10 mM TRIS-Cl (pH 6.5), 0.1 mM EDTA.
TITLE: SARS Direct Saliva Protocol Takarabio
AUTHORS: Timothy Hamerly, Caroline J Stephenson, Borja Lopez-Gutierrez, Rhoel  Dinglasan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Diagnostic testing for SARS-CoV-2 by RT-qPCR has steadily increased since the COVID-19 pandemic began, resulting in the development of protocols for testing from a variety of biological matrices. Initial testing focused on nasopharyngeal and oropharyngeal swabs, which are invasive and require trained health care professionals to perform the sample collection, resulting in a bottleneck in testing. Saliva has emerged as a viable sample specimen for virus detection and can be self-collected with little instruction. Further, it has been demonstrated that virus detection is possible without the need for complex extraction protocols, including commercially available kits. However, a potential pitfall of available kits is the lack of comprehensive testing during development using contrived biological samples containing intact, heat inactivated SARS-CoV-2 virus in the appropriate matrix (e.g., saliva). </div><div class = "text-block">
We have found the Takara Direct One-Step RT-qPCR Mix for SARS-CoV-2 kit to be a cost-effective alternative for testing the direct detection of virus in saliva when compared with magnetic bead-assisted extraction systems, with minimal modifications from the manufacturers protocol. We have found that successful detection of virus requires the addition of a proteinase K digestion step, an increase of the reverse transcriptase time, and 3-step cycle (compared with a 2-step cycle in the manufacturers documentation). These changes allow for the successful detection of SARS-CoV-2 virus with a minimal increase in Ct values for RNase P and viral N-gene transcripts when compared with magnetic bead-assisted extractions kits. Implementing these critical steps will help end-users avoid false negatives. </div></div>

[STEPS]
TITLE: 0.1xBWT+SDS buffer
AUTHORS: Anna Schmidt, Sarah Nagel, Matthias Meyer, Elena Essel

[DESCRIPTION]
Protocol for the preparation of 0.1x BWT+SDS buffer (Bind and wash buffer I) for automated single-stranded DNA library preparation using the ssDNA2.0 method (Gansauge et al. 2020).

References
Gansauge, M.-T., Aximu-Petri, A., Nagel, S., & Meyer, M. (2020). Manual and automated preparation of single-stranded DNA libraries for the sequencing of DNA from ancient biological remains and other sources of highly degraded DNA. Nature Protocols, 15, 2279-2300.

[STEPS]
TITLE: U Mass - Lipase
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">
This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Lipase is a gastrointestinal enzyme that processes fat/lipid digestion.</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 Lipase test on display and run the analysis.
?. Collect and analyze the data.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a protocol from: <br /><br />Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_057.pdf" target="_blank">published manuscript</a> for additional information.

[GUIDELINES]
<strong>Authors: </strong>Kenneth M. Stedman<sup>1</sup>, Kate Porter<sup>2</sup>, and Mike L. Dyall-Smith<sup>3</sup> <br /><sup>1</sup>Department of Biology, Center for Life in Extreme Environments, Portland State University, P.O. Box 751, Portland, OR 97207, USA <br /><sup>2</sup>Biota Holdings Limited, 10/585 Blackburn Road, Notting Hill Victoria 3168, Australia <br /><sup>3</sup>Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany<strong><br /><br /></strong>
<div>Modified growth medium (MGM) contains 5 g peptone and 1 g yeast extract per liter of salt water. The salt concentration is varied according to the host strain, and is detailed in the <a href="http://www.haloarchaea.com/resources/halohandbook/" target="_blank">Halohandbook</a>.</div>

[STEPS]
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TITLE: Bead clean-up (single tube)
AUTHORS: timothy.creed

[DESCRIPTION]
Protocol for purification of DNA using SPRI beads

[STEPS]
SECTION: Prepare reagents
1. Prepare the following reagents/materials:
  or Homemade SPRI beads
Fresh 70% Ethanol
Nuclease-free water or elution buffer
DNA sample/PCR product
Clean 1.5 mL tubes
Magnetic tube rack

 

 

SECTION: Add beads to sample
2. In a 1.5 mL tube, add resuspended beads to sample/PCR product in ratio specified in main protocol. 
 

SECTION: Add beads to sample
2.2. Allow mixture to incubate for 5 min at Room temperature 

 

SECTION: Separation
3. Place tube on a magnetic rack. 
 

SECTION: Add beads to sample
2.1. Flick tube gently to mix beads and sample.
 

SECTION: Separation
3.1. Wait ~1-3 minutes for the beads and buffer to separate.  Beads will stick to magnetic side of the tube and the solution should be clear. 
SECTION: Separation
3.2. Using a pipettor, remove and discard the clear supernatant solution. DNA will remain in the tube bound to the beads.
SECTION: Ethanol Washing
4. Add 500 μL 70% EtOH to tube 
Do not disturb the beads! Pipette into the opposite side of the tube.
 

SECTION: Ethanol Washing
4.1. Incubate at Room temperature  for 30 s 

SECTION: Ethanol Washing
4.2. Using a pipettor, remove and discard the EtOH. Be careful not to disturb or aspirate the beads.
SECTION: Ethanol Washing
5. Repeat the Step 4 EtOH wash. Try to remove as much EtOH as possible without disturbing the beads.
 

SECTION: Air dry beads
6. Leaving tube cap open, allow beads to dry for a maximum of 90 seconds.
 

SECTION: Resuspend
7. Remove tube from magnetic rack and resuspend DNA/beads by adding a minimum of 15 μL nuclease-free water or desired buffer. 
 

SECTION: Incubate
8. Incubate at Room temperature for 2 min- 5 min 
SECTION: Resuspend
7.1. Flick tube gently to resuspend beads in elution.
SECTION: Recovery
9. Place tube back on magnetic rack. Allow beads and elution to separate for 1 min .

SECTION: Recovery
9.1. Slowly aspirate out clear supernatant, now containing DNA, and transfer to final clean container.
TITLE: General freezing protocol for HEK-Blue cells
AUTHORS: Andreas Sagen

[DESCRIPTION]
HEK-Blue is a product from Invivogen, which provide reporter cells for endotoxin-testing among others. Here is a generalized protocol for freezing cells with reporter characteristics. This protocol differ significantly from Invivogens recommendations and should be used with care, as it has not been tested on all the different cell lines provided by Invivogen.

[BEFORE_START]
Prepare freezing medium. Take care to heat and cool the necessary reagents.

[GUIDELINES]
The protocol has been proven to work with different HEK-Blue cells, but not all. Please follow the recommendations of Invivogen when buying a new batch, and test protocol when a sizable batch of HEK-blue cells are available in storage.

[STEPS]
SECTION: Concentrated complete freeze medium (CCMF)
1. Pre-prepare freezing medium. While you could create freeze medium outright, and suspend the cells directly in it, it is recommended to minimize the exposure time of cells with DMSO. To do this, the cells are initially resuspended and counted in serum-free medium. This give you time to create labels, etc. between resuspension and freezing.
SECTION: Concentrated complete freeze medium (CCMF)
1.1. In 70 mL DMEM, mix 20 mL FBS and 10 mL DMSO

 

Materials:
 
 
 
SECTION: Concentrated complete freeze medium (CCMF)
1.2. Aliquot  CCMF in 10 mL units and store frozen at -20 °C

 

SECTION: Freezing procedure
2. When cells reach between 80-90% confluence, the freezing procedure can start

 

 

SECTION: Freezing procedure
2.1. Remove supernatant from monolayer. Store 2 mL or more for mycoplasma testing. Wash cell layer gently with PBS. Add 10 mL PBS to a T-75 equivalent flask, and incubate for 5 min. Lightly tap flask, and ensure cell detachment with microscope

Materials:
 
SECTION: Freezing procedure
2.2. Transfer suspension to a canonical centrifugation tube and centrifuge with 200 rcf, 2 min
SECTION: Freezing procedure
2.3. Decant supernatant and resuspend pellet in 1 mL warm serum-free DMEM

Materials:
 
SECTION: Freezing procedure
2.4. Create a 100 µL 10x dilution of suspension in PBS, and determine cell concentration.
Dilute cell suspension in warm serum-free DMEM to 2×106 live cells mL-1. Find volume DMEM to add with  , where   is the cell concentration found earlier
SECTION: Freezing procedure
2.5. Dispense 500 µL cell suspension to cryotubes or screw cap tubes. Create labels and attach to tubes. Lastly, add 500 µL ice-cold CCFM

 

Mix suspension, and transfer tubes to a cold block (Corning #432050) on ice. Transfer tubes or cold block to an insulated box (Corning #432021). Freeze at -80 °C for 1440 min. Transfer tubes to box, and store long-term in a vapor-phase nitrogen freezer or medium-term in a ultra-low temperature freezer
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Table 1. Antibiotic concentrations used for establishing axenic sea-ice diatom cultures.</p>
<table>
<tbody>
<tr>
<td width="94">
<p><strong>Antibiotic</strong></p>
</td>
<td width="154">
<p><strong>Final concentration (µg/ml)</strong></p>
</td>
</tr>
<tr>
<td width="94">
<p>Ampicillin</p>
</td>
<td width="154">
<p>50</p>
</td>
</tr>
<tr>
<td width="94">
<p>Gentamycin</p>
</td>
<td width="154">
<p>15</p>
</td>
</tr>
<tr>
<td width="94">
<p>Streptomycin</p>
</td>
<td width="154">
<p>125</p>
</td>
</tr>
<tr>
<td width="94">
<p>Chloramphenicol</p>
</td>
<td width="154">
<p>10</p>
</td>
</tr>
<tr>
<td width="94">
<p>Ciprofloxacin</p>
</td>
<td width="154">
<p>10</p>
</td>
</tr>
<tr>
<td width="94">
<p>Penicillin</p>
</td>
<td width="154">
<p>100</p>
</td>
</tr>
</tbody>
</table>
<p> </p>
<p> </p>
<p><strong>References</strong></p>
<p> </p>
<p><strong>Guillard RRL. 1975.</strong> Culture of phytoplankton for feeding marine invertebrates. In: Smith WL, Chanley MH eds. <em>Culture of marine invertebrate animals</em>. New York: Plenum, 29-60.</p>
<p> </p>
<p><strong>Jaeckisch N, Yang I, Wohlrab S, Glöckner G, Kroymann J, Vogel H, Cembella A, John U. 2011.</strong> Comparative Genomic and Transcriptomic Characterization of the Toxigenic Marine Dinoflagellate Alexandrium ostenfeldii. <em>PLoS ONE</em> <strong>6</strong>(12): e28012.</p>

[STEPS]
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TITLE: STC-1 cell culture
AUTHORS: Michael J Hurley

[DESCRIPTION]
Cell culture of STC-1 mouse enteroendocrine cells.

[STEPS]
SECTION: STC-1 cell culture
1. Obtain Mouse (Mus musculus) neuroendocrine duodenal adenoma STC-1 cells (RRID:CVCL_J405) from the American Type Culture Collection (ATCC-CRL-3254™).
SECTION: STC-1 cell culture
2. Grow STC-1 cells in DMEM:F12 Ham with GlutaMax™ media supplemented with 10 % charcoal absorbed foetal bovine serum (LabTech), 1 mM sodium pyruvate, non-essential amino acids (100 mM) and penicillin (100 U/ml) streptomycin (100 mg/ml) on 12 well plates at 37°C under saturating humidity in a 5% CO2/95% air mixture.
SECTION: STC-1 cell culture
3. Subculture cells when 80-90 % confluent using Accutase™ and replate at 1:10 dilution for maintenance or at an appropriate density for experiments.
TITLE: 05 Agarose Gel Electrophoresis
AUTHORS: TJUSLS China

[STEPS]
?. Use 1×TAE buffer to prepare 1% Agarose mix in a flask, then put it in the microwave and heat it as long as it takes to completely dissolve the Agarose.
?. Take out the conical flask, cool it in the wash basin to about 50°C. Add EB  quickly , and then mix well. Pour the Agarose gel into gel tray and insert comb into slots. Let the gel solidify for 15-20min. Meanwhile, dilute the 10x green buffer to 1x and add to the DNA samples. ~
50 °C
?. Place the gel onto the electrophoresis apparatus ensuring that it is totally submerged in 1xTAE buffer. Carefully load each sample into its designated lane and 2ul DNA marker into a separate lane.
2 µl
?. Run at 120V for 20-25 min. If the sample have not completely separated, the time may be extended appropriately. ~
?. Check the gel using a gel imager or under UV light, then take a photo oNorma.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The assay is intended to amplify and sequence the HIV-1 gp120 V3 domain and infer if the virus can use the CXCR4 coreceptor for cell entry. The use of this coreceptor renders the virus not susceptible to the CCR5 coreceptor antagonist maraviroc. </p>

[STEPS]
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TITLE: Bridport Health Liver Support:-SUPPLEMENT FOR LIVER HEALTH
AUTHORS: bridporthealthliver  

[DESCRIPTION]
The body is built to withstand a lot of chaos, but most people take it for granted. Whether drinking alcohol serves as a social lubricant or a symptom of depression, it can lead to addiction for some people. With these years of imbibing, anyone who has spent time in a health class or Alcoholics Anonymous knows the damage that can ensue.

[STEPS]