eamag/protocols-with-steps huggingface dataset:
TITLE: Assembling MAGs using KBase
AUTHORS: John Beckley

[DESCRIPTION]
This protocol describes the steps to run a KBase narrative to assemble MAGs from raw reads.

[STEPS]
1. Create an acount with KBase, login, and start a new narrative.
2. Import metagenomic data as SRA reads by selecting the “Import SRA File as Reads from Web” app. Provide the SRA file URL, the sequencing technology, and an output name for the file to be generated. Run the app and locate output in My Data.
3. To assess quality of the reads, run the “Assess Read Quality with FastQC” app. Load the uploaded data from the SRA upload and run the app.
4. Review the quality scores and determine if any of the reads need to be removed for low quality scores.
5. To trim reads of adapters and to remove low quality reads, load the data into the “Trim Reads with Trimmomatic” app. There are several parameters to adjust, but these can be left at the default unless specific criteria are needed for the analysis. Define an output file name and run the app.
6. Review the quality scores and compare to the pre-trimmed quality report.
7. Prior to assembly, the reads can be used to assign taxonomy. Load the trimmed data file into the “Classify Taxonomy of Metagenomic Reads with Kaiju” app. Select the taxonomic level of classification and the database to be used and run the app.
8. Analyze the data to look at the taxonomic classifications present in the sample.
9. To run the assembly, loaded the trimmed reads into the “Assemble Reads with metaSPAdes” app. Select the minimum contig length, identify an output object, and run the app.
10. Bin the contigs by loading the assembled read object into the “Bin Contigs using MaxBin2” app. Choose an output object and run the app.
11. 10.  Load the result of the binning into the “Assess Genome Quality with CheckM” and run the app.
12. 11.  Analyze and determine which bins are acceptable for extraction. This would be any bins with more than 75% completion and less than 5% contamination.
13. 12.  Extract the selected genomes using the “Extract Bins as Assemblies from BinnedContigs”.
14. 13.  Metagenome assembled genomes are now ready to be annotated and further analyzed in KBase, or exported for analysis by other methods.
TITLE: Transplantation of Chicken Egg-White Extract-Induced Rabbit PBMCs as a Treatment for Renal Ischemia-Reperfusion Injury in Rabbits
AUTHORS: ruangp 

[STEPS]
?. Identification of noninduced and induced PBMCs
?. Preparation of the rabbit renal ischemia-reperfusion injury model
?. Rabbit PBMC isolation, culture, induction and labeling
?. Kidney function test
?. Changes in the urinary protein content
?. Observation of labeled cells in frozen sections
?. Histopathological assessment of the kidneys
?. Metabolomics analysis of the kidney
?. Statistical analysis
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for human small intestine cell dissociation, performed on ice to reduce artifact gene expression. </p>

[BEFORE_START]
<div title="Page 1">
<p><strong>Checklist prior to beginning:</strong><br />-Centrifuges, large and small, set to 4 C<br />-Make enzyme stock; place 2 tubes of enzyme on dry ice.<br />-Make 0.01% BSA/PBS (50 mL)<br />-Things you need: petri dishes, clean forceps, razor blade, pipets, 30 μM filters, timer.</p>
<p> </p>
<p><strong>Stock solution for enzyme </strong></p>
<ul>
<li>895 μL DPBS</li>
<li>5 μL 0.5 M EDTA (2.5 mM final)</li>
</ul>
<p>→Add 100 μL enzyme (100 mg/mL) to 900 μL of enzyme stock to make 1X enzyme mix. Add 28 mg of tissue to each 900 μL of enzyme mix.</p>
</div>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>MASAM MASAM is a useful tool discerning between memories of self-experienced real - life events and fabricated or fictitious accounts.<br />The theoretical framework of MASAM are four assumptions. First, it is necessary to consider the fact that in each statement, truthful anddeceptive, true and false information can be found. Moreover, if a witness’ intention is to give untruthful picture of actual events, certain differences appear in form and content of the given statement; the truthful parts will differ from lies. Furthermore, it is necessary to refer to the course of statement formation, that is to analyze circumstances of the event (what happened?), to establish witness’ characteristics (who gave the statement?) and to assess the course of the interview (how was the witness interviewed?). In accordance to MASAM assumptions high evidential value of a statement is based on and proven (supported?) by coherence between (1) the content and form of a statement, (2) the object and event features (i.e. complexity and observation conditions), (3) observer's characteristics (i.e. witness' prior experiences, age) and (4) interviewing circumstances (i.e. how detailed was the interview, when was the witness interviewed). MASAM does not assume that in witness's statement which describes one's experiences, certain elements should be found.<br />There are 21 MASAM content criteria, divided into three categories: general features, details and deposition. Each of the content criteria is assessed separately with respect to object and event features (what happened?), observer's characteristics (who is the suspect?) and interviewing circumstances (how was the witness questioned?). Verbal cues are supposed to assist analysis of respective areas related to formation of statements as well as to be a kind of a guide to direct attention to areas that require more detailed analysis.</p>

[STEPS]
?.
TITLE: Mask-Based Covid-19 testing system using Exhaled Breath Condensate
AUTHORS: John Daniels

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The DiagMetrics (fka Kinaptic) mask-based COVID-19 testing system uses a unique Exhaled Breath Collector (EBC) that converts breath to a liquid bio sample (condensate) for rapid testing and wireless automatic contact tracing. The Exhaled Breath Condensate is collected from a fan-shaped condenser embedded in a mask, which then funnels the sample to our proprietary electronic biosensor. The biosensor is functionalized using engineered Covid-19 antibody fragments as the capture molecules.  These engineered antibody fragments have a strong and selective affinity for the COVID-19 S-protein.  Once the S-protein attaches to the antibody fragments, the electrical characteristics of the biosensor change, resulting in a change in current flowing through the biosensor.  The change in current triggers a signal that is sent via Bluetooth to a smart phone app, which then signals to the user a “positive” test for COVID-19.  This smart phone app can then be used to perform contact tracing.  If the change in current is below a predetermined threshold, the test will be deemed “negative".  In the situation where a test is “negative", the full testing system can be reused.  Our electronic biosensor has been proven to detect low concentration levels of the COVID-19 S-protein and typically takes less than 10 minutes for a test to be completed.</div><div class = "text-block">The DiagMetrics mask-based testing system is low cost, easy to use, and provides rapid results.  This testing system can be used at home, in businesses, in schools or in other municipal and public settings to provide a method for proactively detecting and isolating positive Covid-19 cases before the virus is transmitted to other people.  </div></div>

[STEPS]
?. Remove release liner from double stick tape on electronics
?. Remove release liner from double sided tape on mask
?. Pull tab to Activate Battery and Adhere electonics to mask
?. Open APP on Smartphone
?. Connect to Mask Electronics
?. Put mask on and breath normally.
?. Continue to breath normally
?. Testing is Complete
?. Upload results
TITLE: Relative viscosity
AUTHORS: Alison Lovegrove

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lab protocol for: Relative viscosity measurement in wheat flour using a capillary viscometer.</div><div class = "text-block"><span>based on Saulnier et al. (1995) Journal of Cereal Science, </span><span style = "font-weight:bold;">22</span><span>, 259-264</span></div></div>

[STEPS]
TITLE: Protocol for microCT inspection of Gallium particle accelerator targets
AUTHORS: Muofhe Tshibalanganda, Anton du Plessis, Stephan le Roux, Paul Papka

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol described the steps used to inspect the Gallium targets used in particle accelerators. These targets are metal disks filled with Gallium, and laser welded on the edges. Various production quality issues may be present - the microCT process needs to be a standard, routine methodology for direct comparison among all such data sets. This protocol sets out the exact steps used for this analysis type.</div></div>

[STEPS]
?. [Sample Mounting]
Load the sample on a foam (e.g. florist oasis) with prestik at top, but angled towards the flat side by up to 10 degrees. This is to reduce penetration artefacts - perfect vertical is not suggested.
?. [Scanning settings]
This is using a General Electric VTomex L240 system, but most microCT systems would work with similar settings.  AB1Voltage200 kV2Current150 μA3X-ray Sensitivity44Beam
  Filter&nbsp;0.5 mm copper5Detector
  filter&nbsp;0.5 mm copper plate6Voxel size30 µm7Timing333 m/s8Images28009Averaging1
AB1Voltage200 kV2Current150 μA3X-ray Sensitivity44Beam
  Filter&nbsp;0.5 mm copper5Detector
  filter&nbsp;0.5 mm copper plate6Voxel size30 µm7Timing333 m/s8Images28009Averaging1
?. [Reconstruction]
Reconstruction of the 3D data is done using General Electric Datos 2.2, using specifically beam hardening correction value of 9.3 - this reduces beam hardening artefacts significantly.
?. [Analysis]
1. Analysis of 3D data is described here using Volume Graphics VGSTUDIO MAX 3.2 . 2. Register the sample - use simple registration to turn the sample upright in the software windows.3. De-noising is done using the filters Adaptive Gauss with smoothing factor 2, edge threshold=0.2, iteration=3.
?. [Animation]
Create Vvdeos of only front and rotation slice with 5mm per front slice and 0.1 degree per rotational slice. Video format: .wmv with naming: Batch # Target # Front slice Batch # Target # Front slice 90 degree rotation Batch # Target # Rotation Batch # Target # Rotation 90 degree rotation- Rotation video are started from west (artefacts at North and South) and rotated anticlockise. Capsule is facing up, welded plate is facing downFront slice videoRotation video
?. [Saving]
The data is saved including the filtered data set, as a image stack (tiff). These can be viewed manually without special software. The analyzed volume can be opened in free myVGL software.
?. [Sample loading]
Mark the top of the disk with a small ball of prestik, this is the reference for orientation to check second scan rotation by 90 degrees (eg. in data set after scanning, to ensure the disk was rotated).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Experiment purpose is to monitor the time-course of a large-scale infection of host cyanobacteria by phage under variable media conditions and obtain samples for proteomic and transcriptomic analysis.</strong></p>
<p> </p>
<p><strong>8 Hourly Timepoints: 0, 2, 4, 6, 8, 10, 12, 14</strong></p>
<p> </p>
<p><em>Sampling is to determine total cell concentration by flow cytometry.</em></p>
<p> </p>
<ul>
<li><u>For Flow cytometry</u>, <strong>100 µl of sample in duplicate</strong> were taken from each experiment bottle at every other time-point and diluted 10 X in ASW salts, followed by fixing the sample by adding 5 µl of 25% glutaraldehyde, incubating in dark for about 10 minutes, and then flash freezing in liquid nitrogen. Samples were stored at -80°C for further processing.</li>
</ul>

[STEPS]
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TITLE: Quantitative PCR, 384 well format
AUTHORS: Malu G Tansey

[DESCRIPTION]
Quantitative PCR, 384 well format

[STEPS]
SECTION: Procedure:
2. Place 8-tube PCR strips in PCR tube racks (each single tube runs 1 sample and 1 gene)
SECTION: Procedure:
3. Add 5 µl cDNA to the bottom of each tube, use 20 uL pipetor (keep on ice)
SECTION: Procedure:
4. Add 25 µl (duplicates) or 35 µl (triplicates) of Sybr Mastermix to the 8-well tube (keep on ice)
SECTION: Procedure:
4.1. Use 200uL multi-channel pipette.
SECTION: Procedure:
5. Mix using the multichannel and quick spin.
SECTION: Procedure:
6. Dispense 10 µl of cDNA/Mastermix (20uL pipetor) into each well on 384 well plate according to plate layout made in advance (keep plate on ice)
SECTION: Procedure:
7. Gently blot top of plate with kimwipe (to keep samples from transferring to other wells)
SECTION: Procedure:
8. Place clear Adhesive plate cover over the plate.
SECTION: Procedure:
8.1. use brown ‘helper’ to smooth out
SECTION: Procedure:
8.2. pay attention to edges
SECTION: Procedure:
8.3. work from center of the plate out
SECTION: Procedure:
9. Spin plate for 5 min at 3500 rpm (4° C)
SECTION: Procedure:
9.1. During spin: set up ABI SDS program (keep plate in centrifuge until ready to run)
SECTION: Procedure:
10. Seal plate with sticky film. Vortex and spin down plate 3500rpm for 5 min at 4C
SECTION: Procedure:
11. Open SDS 2.3 program
SECTION: Procedure:
11.1. File -> new
SECTION: Procedure:
11.2. One instrument tab: real time -> Connect to machine -> open/close door
SECTION: Procedure:
11.3.  Insert plate, aligning A1 to A1
SECTION: Procedure:
11.4. Close door
SECTION: Procedure:
11.5. On layout tab; highlight unused wells, click “omit wells”
SECTION: Procedure:
11.6. Highlight used wells and click “add detector” for each specific gene
SECTION: Procedure:
11.7. Set to 10uL Rxn VL
SECTION: Procedure:
11.8. Check cycle times and temperatures
SECTION: Procedure:
11.9. Add dissociation stage (SYBR primers only)             

 

SECTION: Procedure:
12. Run plate     
SECTION: Procedure:
13. Primer Validation Procedure: Set-up is same as above plus cDNA standard curve for each gene in an extra set of 8-tube PCR strips (see workflow file)
SECTION: Set-up:
1. Sample cDNA:  thaw your cDNA on ice (vortex and quick spin before plating)
SECTION: Set-up:
1.1.         Primer stock (+/-): Add 12.5 µl of (+) and 12.5 µl (-) to 1 mL MilliQ H2O
SECTION: Set-up:
1.2.   PCR Master Mix (10ul rxn; Triplicate)
TITLE: 0.1M Phosphate Buffer
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol is used to prepare 0.1M Phosphate Buffer. 0.1M Phosphate Buffer is used in the preparation of Sucrose solutions for cryoprotection.

Note: 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.

[STEPS]
TITLE: Small-scale silencing experiment in vegetative Euplotes crassus (provisional)
AUTHORS: Rachele Cesaroni

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Grow RNAse III deficient E.coli strain HT115 in LB with antibiotic selection o/n at 37°C.
?. Prepare a 1:100 dilution of the bacterial culture, and grow it at 37°C until it reaches an OD600 of 0.4.
?. Add 0.4 mM IPTG, and induce RNA transcription from the L4440 plasmid in the bacteria o/n at 37°C.
?. Collect the bacteria by centrifugation at max speed for 10 minutes, and wash them once with ddH2O.
?. Isolate at least 30 well-starved Euplotes crassus cells, and feed them twice a day with feeding bacteria.
?. Leave the Euplotes crassus cells at 24°C, and monitor daily the change in phenotype.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Introduction</strong></p>
<p>Despite relevant evidence that supplemental oxygen therapy can be harmful to patients with myocardial injury, the association between hyperoxia and the clinical outcome of such patients has not been evaluated. We assessed whether early hyperoxia negatively affects outcomes in hospitalized patients with myocardial injury.</p>
<p><strong>Methods</strong></p>
<p>This was a retrospective study conducted at a tertiary referral teaching hospital. Between January 2010 and December 2016, 2,376 consecutive emergency department patients with myocardial injury, defined as a peak troponin-I level ≥ 0.2 ng/mL, within the first 24 hours of presentation were included. The metrics used to define hyperoxia were the maximum average partial pressure of oxygen (PaO<sub>2MAX</sub>), average partial pressure of oxygen (PaO<sub>2AVG</sub>), and area under the curve during the first 24 hours (AUC<sub>24</sub>). The association between early hyperoxia within 24 hours after presentation and clinical outcomes was evaluated using multiple imputation and logistic regression analysis. The primary outcome was 28-day in-hospital mortality. The secondary outcomes were new-onset cardiovascular, coagulation, hepatic, renal, and respiratory dysfunctions (sequential organ failure sub-score ≥ 2).</p>
<p><strong>Results</strong></p>
<p>Compared with normoxic patients, the adjusted odds ratios (ORs) for PaO<sub>2MAX</sub>, PaO<sub>2AVG</sub>, and AUC<sub>24</sub> were 1.55 (95% confidence interval (CI) 1.05–2.27; p = 0.026), 2.13 (95% CI 1.45–3.12; p = 0.001), and 1.73 (95% CI 1.15–2.61; p = 0.008), respectively, in patients with mild hyperoxia and 6.01 (95% CI 3.98–9.07; p &lt; 0.001), 8.92 (95% CI 3.33–23.88; p &lt; 0.001), and 7.32 (95% CI 2.72–19.70; p = 0.001), respectively, in patients with severe hyperoxia. The incidence of coagulation and hepatic dysfunction (sequential organ failure sub-score ≥ 2) was significantly higher in the mild and severe hyperoxia group.</p>
<p><strong>Conclusions</strong></p>
<p>Hyperoxia during the first 24 hours of presentation is associated with an increased 28-day in-hospital mortality rate and risks of coagulation and hepatic dysfunction in patients with myocardial injury.</p>

[STEPS]
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TITLE: Stereotaxic α-Syn PFF injection into mouse striatum
AUTHORS: Ramhari Kumbhar, Hanseok Ko, Valina L. Dawson, Ted Dawson, Xiaobo Mao

[DESCRIPTION]
For stereotaxic injection of α-Syn PFF into the mouse striatum using 2-3 month old mice. This forked protocol has slight variations in anesthesia solution and injection coordinations.

[BEFORE_START]
Personnel should use aseptic techniques and conduct the surgery using sterile gloves in a sterile environment. Surgical instruments should be sterilized before surgeries using an autoclave and during the procedure using glass bead
sterilizer.

[STEPS]
1. Anesthetize mice with a ketamine cocktail (ketamine 100mg/kg; xylazine 10mg/kg; i.p. or s.c.).
2. Shave the animals’ skulls. 
3. Create a 1.5 cm incision in the scalp after cleaning the scalp with alcohol or betadine swabs.  
4. Retract the underlying fascia will and drill a hole in the skull.  
5. Insert a 26 gauge needle attached to a 2uL Hamilton syringe containing recombinant α-syn PFF (5 μg/2 μL) or an equivalent volume of PBS into the striatum unilaterally (right hemisphere) at coordinates
mediolateral 2.0 mm from bregma, anteroposterior -0.2 mm, and dorsoventral 2.8mm. at a rate of 0.4 ul per minute using a minisyringe pump (Model BS-800, Braintree Scientific). 
6.     
Leave the needle in place for 5 minutes before withdrawing the needle. 
7.     
Close the incision using sterile wound healing clips (7 mm stainless steel wound clip; Reflex 7 skin closure system) or sutures. 
8. Immediately after the stereotaxic procedure, treat the stapled wound site with bupivacaine (0.4-0.8 ml/kg of a 0.25% solution, injected sc) or 0.5% xylocaine 1.4 ml/kg, injected sc, injected) or 0.5% xylocaine when necessary or as determined by animal use policy.
TITLE: Water filtration with Zymo Bashing Bead tubes and DNA/RNA Shield reagent for subsequent DNA and RNA extraction from microbial communities
AUTHORS: Christopher A Hempel

[DESCRIPTION]
Filtration of water samples with Zymo Bashing Bead tubes and DNA/RNA Shield reagent for subsequent DNA and RNA extraction from microbial communities.

[BEFORE_START]
Label bashing tubes (1 per sample + 1 filtration control) and load them with 1 mL of DNA/RNA Shield

[STEPS]
SECTION: Procedure
1. Sterilize forceps and scissors with burner, then bleach, then EtOH before each filtration
SECTION: Procedure
2. Filter water sample, make sure to keep the filtration unit closed when opening the wrapping, to hold nothing but the sample over the unit, and to stand as far as possible away from the unit while it is open
SECTION: Procedure
3. Cut filter into small pieces (forceps and scissors) while filter lays on the filtration unit, make sure to stand as far as possible away from the unit
SECTION: Procedure
4. Transfer pieces into bashing tubes until all pieces are covered with DNA/RNA Shield
SECTION: Procedure
5. Clean filtration unit adapter with bleach after each filtration
SECTION: Procedure
6. Bashing tubes can be stored at -20°C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p style="margin: 0in 0in 0pt; line-height: normal;"><span style="color: #3c3c39; font-family: 'Arial',sans-serif; font-size: 10pt;">This is a modified version of the DNA extraction methods published in:<br /> Rowan, R., &amp; Powers, D.A. (1991) Molecular genetic identification of symbiotic dinoflagellates (zooxanthellae). <em><span style="font-family: 'Arial',sans-serif;">Marine Ecology Progress Series</span></em>, <em><span style="font-family: 'Arial',sans-serif;">71</span></em>(1), 65–73.<br /> Baker, A.C., Rowan, R., &amp; Knowlton, N. (1997) Symbiosis ecology of two Caribbean acroporid corals. <em><span style="font-family: 'Arial',sans-serif;">Proceedings of the 8<sup>th</sup> International Coral Reef Symposium, Panama </span></em>2: 1295-1300<br /> <br /> This protocol is used to extract and purify genomic DNA from coral samples (tissue and skeleton) that is suitable for PCR, qPCR, sequencing, and other downstream applications. DNA is recovered from the coral host, <em>Symbiodinium</em>, and other members of the holobiont.</span></p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This brief protocol shows you how easy it is to get shRNA from demostrated siRNA sequence. With these self-designed shRNA, I've got more than 10 genes knockdown from mammalian cell lines, including mouse embryonic stem cells. Believe it or not, using siRNA sequence from Dharmacon will guarantee you the success.

[STEPS]
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TITLE: Statistical analysis
AUTHORS: carmela.giachino

[DESCRIPTION]
Statistics 


[STEPS]
SECTION: Statistical Analyses
1. Statistics was carried out using GraphPad Prism Software Version 9 (GraphPad Software, San Diego, California, USA). Significance was calculated using the unpaired, two-tailed Student’s t-test; Two-way ANOVA, followed by Bonferroni-post hoc test, as appropriate.
TITLE: DNA extraction from frozen whole blood using the DNeasy Blood & Tissue kit (QIAGEN)
AUTHORS: Luise A. Seeker, Jennifer Fairlie, Sarah L. Underwood, Rachael V. Wilbourn, Rebecca Holland, Daniel H. Nussey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Even though DNA extraction from whole blood is a standard protocol of most commercial kits DNA quality and purity can be disappointing if those protocols are not optimised. Here, we present an optimised protocol for the extraction of high quality genomic DNA using the DNeasy Blood & Tissue kit (QIAGEN). Our downstream application of the DNA was the measurement of relative leukocyte telomere length by qPCR. The protocol might also be appropriate for other downstream applications. Changes reported here significantly improved DNA yield and 260/280 and 260/230 ratios measured on NanoDrop. </div></div>

[STEPS]
TITLE: SMARTerV4 (1x) Amplification for single-cell or  single-nuclei RNASeq Protocol
AUTHORS: ZengU19 BRAIN grant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol to generate full-length cDNA from single cells, or nuclei, using Takara SMARTer V4.</div></div>

[STEPS]