eamag/protocols-with-steps huggingface dataset:
TITLE: DNA extraction from fecal samples
AUTHORS: Yoshiyuki Matsuo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">DNA extraction from fecal samples</div></div>

[STEPS]
?. [Materials]
Phosphate-buffered saline (PBS)EZ-beads (Promega, AMR76813M): 2 mL tube containing 0.2 mm ceramic (zirconium dioxide) spheres and one 5 mm zirconia beadMaxwell RSC Blood DNA Kit (Promega, AS1400)MN Bead Tube Holder (MACHEREY-NAGEL, 740469): Rubber-foam adapter for processing bead tubes with Vortex-Genie instrument
?. [Instrument]
Maxwell RSC (Promega, AS4500): Automated nucleic acid purification platformMicro Smash (TOMY, MS-100): Beads cell disrupterVortex-Genie MixerHeating blockMicrocentrifuge
?. [Preparation of fecal samples]
Place 50-100 mg of fecal sample into tube.
?. [Preparation of fecal samples]
Add 1 ml of PBS per 100 mg of feces.
?. [Preparation of fecal samples]
Mix thoroughly by vortexing or pipetting.
?. [Preparation of fecal samples]
Allow the sample to stand for 2 min to sediment large debris.
?. [Preparation of fecal samples]
Transfer 300 µl of the suspension to 1.5 mL tube.
?. [Preparation of fecal samples]
Centrifuge at 18,000 x g for 3 min.
?. [Preparation of fecal samples]
Discard the supernatant.
?. [Preparation of fecal samples]
Resuspend the pellet (~30 mg of feces) in 300 µl of PBS.
?. [Preparation of fecal samples]
Incubate at 70°C for 10 min and cool to room temperature.
?. [Mechanical cell disruption by beat beating]
Transfer 300 µl of the suspension to EZ-beads tube.
?. [Mechanical cell disruption by beat beating]
Lyse cells either by using disruption device (13.1) or vortex mixer (13.2).
?. [Mechanical cell disruption by beat beating]
Place the EZ-beads tube in Micro Smash instrument and disrupt cells at 2,500 rpm for 2 min.
?. [Mechanical cell disruption by beat beating]
Place the EZ-beads tube on MN Bead Tube Holder attached to Vortex-Genie mixer and vortex for 5 min at maximum speed.
?. [Mechanical cell disruption by beat beating]
Briefly spin the tube.
?. [Automated DNA extraction using Maxwell RSC Blood DNA Kit]
Add 300 µl of Lysis Buffer and 30 µl of Proteinase K Solution to the sample in EZ-beads tube.
?. [Automated DNA extraction using Maxwell RSC Blood DNA Kit]
Mix thoroughly by vortexing for 10 sec.
?. [Automated DNA extraction using Maxwell RSC Blood DNA Kit]
Incubate at 56°C for 20 min.
?. [Automated DNA extraction using Maxwell RSC Blood DNA Kit]
Briefly spin the tube.
?. [Automated DNA extraction using Maxwell RSC Blood DNA Kit]
Transfer the supernatant (~600 µl) to 1.5 mL tube.
?. [Automated DNA extraction using Maxwell RSC Blood DNA Kit]
Centrifuge at 18,000 x g for 3 min.
?. [Automated DNA extraction using Maxwell RSC Blood DNA Kit]
Transfer the cleared lysate to Maxwell RSC Cartridge.
?. [Automated DNA extraction using Maxwell RSC Blood DNA Kit]
Add 50 µl of Elution Buffer to elution tube.
?. [Automated DNA extraction using Maxwell RSC Blood DNA Kit]
Start the extraction run following the manufacturer's instructions.
TITLE: Collection and  Post-Surgical Excision of Human Kidney Tissue through the Cooperative Human Tissue Network
AUTHORS: Maya Brewer, Jamie Allen, Carrie  Romer, Elizabeth Neumann, Agnes Fogo, Raymond Harris, Danielle Gutierrez, Mark De Caestecker, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">Obtain kidney tissue and metadata about tissue location within the whole kidney for storage and analysis.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">A portion of kidney tissue and a series of images that provide information about the original location of the smaller tissue.</div></div>

[STEPS]
?. Kidney tissues are selected based on patient and clinical metadata (e.g. age
?. Receive excised kidney from full nephrectomy.
?. Cut kidney in half along the longest plane and open like a book.
?. Cut out a portion of the kidney (~80 x 60 x 4 mm) that is farthest away from the tumor location.
?. Take a picture of the smaller piece of kidney in reference to its original location within the entire kidney if possible.
?. Cut the piece of kidney into ~4 smaller, rectangular pieces (~2 x 15 x 4 mm).
?. Image each of these smaller pieces in reference to their original position.
?. Place tissue in plastic container on ice.  Transfer tissue to those preparing tissue for long-term storage.
TITLE: Qaigen RNEasy RNA extration protocol
AUTHORS: Jessica Pardy, Michael Dan Siemon, Dilan Joseph, Justin Donovan, Richard Gibson, christopher.degroot, Amanda Hamilton

[DESCRIPTION]
This protocol was employed by Western University for RNA extraction of wastewater samples for wastewater-based epimelogy in London, Ontario, Canada and surrounding area. The protocol was adapted from QIAamp® Viral RNA
Mini Handbook for use with the Qiagen RNeasy extraction kit.

[STEPS]
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
1. Thoroughly mix wastewater sample then aliquot 40 mL into 50 mL Falcon tube. Centrifuge at 12000 RPM for 90 min. Decant supernatant, assume 280 µl pellet.
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
2. Pipet 1120 µl prepared Buffer AVL into Falcon tube with sample
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
3. Pulse-vortex samples for 15s every 2 minutes for 8 minutes
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
4. Add 1120ul ethanol then centrifuge at 4000RPM for 5 minutes
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
5. Process supernatant through QiAmp Mini column by adding 750 µl and centrifuging at 8000 rpm for 1 min. Remove flow-through and repeat until all supernatant has been processed
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
6. Add 500 µl Buffer AW1 to QiAmp Mini column and centrifuge at 6000 x g (8000 rpm) for 1 min. Replace the collection tube with a clean 2 ml collection tube
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
7. Add 500 µl Buffer AW2 to QiAmp Mini column and centrifuge at 6000 x g (8000 rpm) for 1 min. Replace the collection tube with a clean 2 ml collection tube
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
8. Dry membrane by centrifuging at 13000 rpm for 3 min
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
9. Place the QIAamp Mini column in a clean 1.5 ml eppendorf tube.
Add 60 µl Buffer AVE equilibrated to room temperature.
SECTION: Protocol modified from QIAamp® Viral RNA Mini Handbook
10. Centrifuge at 6000 x g (8000 rpm) for 1 min
TITLE: Mouse Perfusion Protocol
AUTHORS: Michael Lee

[DESCRIPTION]
This protocol details the mouse perfusion procedure.

[STEPS]
SECTION: For perfusion with 1x K+-free PBS:
1. Place mouse in isoflurane chamber until unresponsive to toe pinch stimuli.
SECTION: For perfusion with 1x K+-free PBS:
2. Proceed to cut abdominal skin down both sides to expose the diaphragm.
SECTION: For perfusion with 1x K+-free PBS:
3. Cut around diaphragm to expose the heart.
SECTION: For perfusion with 1x K+-free PBS:
4. Cut right atria and insert perfusion needle into left ventricle with pump set to 5 rpm.

SECTION: For perfusion with 1x K+-free PBS:
5. Perfuse 80 mL - 100 mL K+-free PBS until the liver is clear and liquid expelling from heart is completely clear.

SECTION: For perfusion with 1x K+-free PBS:
6. Carefully remove brain and cut on mid-sagittal line using brain matrix.
SECTION: For perfusion with 1x K+-free PBS:
7. Drop-fix left hemisphere in cold 4% paraformaldehyde for histological processing.
SECTION: For perfusion with 1x K+-free PBS:
8. Sub-dissect right hemisphere for regions of interest.
TITLE: Collection of protocols for cell-free protein synthesis
AUTHORS: Weston Kightlinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the collection of protocols for cell-free protein synthesis.</div><div class = "text-block"><span>Read more on cell-free protein synthesis here: </span><a href="https://www.swiftscalebio.com/blog/cell-free-protein-synthesis" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.swiftscalebio.com/blog/cell-free-protein-synthesis</span></a></div></div>

[STEPS]
TITLE: UNC Chapel Hill Zebrafish Aquaculture Core (ZAC) Environmental Summary
AUTHORS: Michelle Altemara

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Zebrafish (Danio rerio) are a popular animal model used in a variety of research areas. As with all animal models, husbandry conditions, including environmental parameters, nutrition, and exposure to pathogens can affect research results. Additionly, zebrafish are tolerant of a wide range of environmental parameters, which has led to wide variation in husbandry practices across facilities around the world. The lack of standard conditions across zebrafish laboratories may play a role in irreproducible experiments. Therefore, annual summaries of these conditions have been compiled for the Zebrafish Aquaculture Core (ZAC) facility at the University of North Carolina (UNC) Chapel Hill with the aim to have this data included in research articles published using our fish. As more institutions publish husbandry protocols, standard husbandry practices may emerge.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p style="text-align: center;"><img style="display: block; margin-left: auto; margin-right: auto;" src="https://s3.amazonaws.com/pr-journal/hnnfjtn.png" width="100" height="90" data-src="https://s3.amazonaws.com/pr-journal/hnnfjtn.png" data-ofn="nehirarma-üst-Tr.png" />  </p>
<p style="text-align: center;">Nehir Biyoteknoloji Ltd. <a href="http://www.nehirbt.com.tr/en/" target="_blank">www.nehirbt.com</a></p>

[BEFORE_START]
<p>This protocol is also called "soft lithography" in the literature. </p>
<p> </p>
<p>The PDMS material is Sylgard 184 silicone elastomer, Dow-Corning.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: SAPAP3 Genotyping Protocol
AUTHORS: kajsbr  

[DESCRIPTION]
Protocol for SAPAP3 KO Mouse Model Genotyping from Dr. Lisa Gunaydin's Lab, University of California, San Francisco

[STEPS]
1. Cut the tails. 
To cut the tails and mark the animals, keep in mind to:
● cut as little amount of the tail as possible;
● always clean the scissors properly in between animals (EtOH and bleach);
● tattoo the animals at the same time as cutting the tails;
● use iso if needed.
2. Digest the tails.
Incubate the tails O/N at 55 C (water bath) in 200 ul 1x PBND buffer with Proteinase K at 0.25 mg/ml. Vortex before.
200ul * (54+1) = 11000ul --> 11ml PBND
0.25mg/ml * 11ml = 2.75mg --> 0.00275g Proteinase K
PBND (250 ml 1x, stored at 4 C)
2.5 ml 1M TrisHCl pH 8.3
1.125 ml IGEPAL
1.125 ml Tween 20
0.935 g KCl
0.125 g MgCl2.6H20
0.025 g Gelatin
Fill remainder with H2O
*Gelatin will dissolve only after heated
3. Inactivate Proteinase K.
After taking the tails out of the bath, see if they are digested. If so, vortex and boil them for 10 min. Vortex again and centrifuge at max speed for 5 min (table top centrifuge, something like 14,000 rpm).

5. Run the 1% agarose gel
200 ml TAE
2g agarose
2ul sybr safe gel stain
24ul DNA per well. 5ul DNA ladder before each new line
4. PCR reaction.
We use the KAPABiosystems HotStart ReadyMix (2x) which contains DNA Polymerase, rxn buffer, dNTPs and MgCl2.
Pipette 24 ul of Master Mix into each PCR tube
Add 1 ul of DNA from each sample into each tube


Sap3 KO F1, Forward primer, 5' - 3': ATTGGTAGGCAATACCAACAGG 
Sap3 KO R1, Reverse Primer 5' - 3': GCAAAGGCTCTTCATATTGTTGG 
Extra Primer, TKF2: CTTTGTGGTTCTAAGTACTGTGG


1 rxnMM SAPAP (34 rxns)Primer nameTotal RxnsForward primer134Sap3 KO F134Reverse primer134Sap3 KO R1Extra primer134TKF2Rxn mix12.5425H208.5289DNA 1.0  PCR programSap3

PCR Program for SAPAP3 Genotyping: 
1. 95C for 4 minutes
2. 30 times: 
    94C for 30 seconds
    62C for 30 seconds
    72C for 60 seconds
3. 72C for 5 minutes 
4. 4C hold

Expected band lengths: 
WT: one band at ~147 bp
Het: one band at ~147 bp and one band at ~222 bp
KO: one band at ~222 bp
TITLE: Fluorescence size exclusion chromatography (FSEC) of native nanodiscs 
AUTHORS: Caroline Brown, Snehasish Ghosh, Kallol Gupta

[DESCRIPTION]
This is a protocol describing how to conduct Fluorescence size exclusion chromatography of native nanodiscs.

[STEPS]
1. Conduct extraction into native nanodiscs following established protocols.
2. Inject 200uL native nanodiscs onto a Superose6 (10/300GL) column which is pre-equilibrated with TBS.
3. Perform separation at a flow rate of 0.5 mL/min and detect eluent with a Shimadzu fluorometer with excitation set to 488 nm and emission set to 509 nm.
4. Collect eluent for downstream analysis.
TITLE: Do differences exist in the hamstring muscles architectural characteristics of elite-level male and female rugby players
AUTHORS: Kevin Cronin, Shane Foley, Sean Cournane, Eamonn Delahunt

[DESCRIPTION]
Hamstring injuries carry a high injury burden and are more prevalent in males than females. This study is designed as a cross-sectional study with two groups. Forty elite rugby union players (20 males; 20 females) will have their hamstring muscle architecture (fascicle length, pennation angle and muscle thickness) measured using B-mode ultrasound. Muscle architecture is a modifiable risk factor associated with hamstring injuries. The aim of this study will be to determine whether differences exist in the hamstring muscles (Bicep Femoris long head, Bicep Femoris short head, Semitendinosus and Semimembranosus) architectural characteristics of elite- level male and female rugby union players.

[STEPS]
TITLE: Statistical Analysis Plan (Part 8 of &#34;Effects of Online Exercise Intervention on Physical and Mental Conditions in Young Adults with Chronic Neck Pain&#34;)
AUTHORS: Yiting Lin

[DESCRIPTION]
This is Part 8 of "Effects of Online Exercise Intervention on Physical and Mental Conditions in Young Adults with Chronic Neck Pain".
Continuous data were expressed as means and standard deviations. Categorical data were shown as frequencies or percentages. All statistical analyses were conducted using theIBM SPSS Statistics for Windows, Version 24.0 (Armonk, NY: IBM Corp). The normality of continuous data was checked with the Shapiro-Wilk test.The change scores of the primary and secondary outcome variables from baseline to end of intervention between the two groups were compared using independent sample t test. Within-group comparisons were done using paired sample t test.

[GUIDELINES]
Statistical Analysis Plan:

Continuous data were expressed as means and standard deviations. Categorical data were shown as frequencies or percentages. All statistical analyses were conducted using the IBM SPSS Statistics for Windows, Version 24.0 (Armonk, NY: IBM Corp). The normality of continuous data was checked with the Shapiro-Wilk test. The change scores of the primary and secondary outcome variables from baseline to end of intervention between the two groups were compared using independent sample t test. Within-group comparisons were done using paired sample t test. The level of significance of all statistical tests was set with two-tailed at 0.05. Per-protocol analysis was used. The sample size for the study was calculated to be 32 using the G*Power 3.1.9.2 (Universitat Kiel, Germany), with an estimated effect size of group difference of 0.9 in primary outcomes and a level of significance of 0.05, statistical power of 80% with two-tailed independent sample t test. The estimated effect size was based on previous studies of home exercise on patients with neck pain (Zronek et al., 2016). With an anticipated dropout rate of 15%, a total of 39 participants was required.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Assembling <em>actual</em> reads from the <a href="https://github.com/MicroB3-IS/osd-analysis" target="_blank">Ocean Sampling Day (2014)</a> using SPAdes, an assembler implemented in <a href="cyverse.org" target="_blank">Cyverse</a>.

[BEFORE_START]
To run this protocol, users must first <a href="https://user.iplantcollaborative.org/register/" target="_blank">register</a> for Cyverse account. All data (both inputs and outputs) are available within Cyverse's data store at /iplant/home/shared/iVirus/ExampleData/

[GUIDELINES]
<p>This is part of a larger protocol <em>Collection</em> that involves the end-to-end processing of raw viral metagenomic reads obtained from a sequencing facility to assembly and analysis using Apps (i.e. tools) developed by iVirus and implemented within the Cyverse cyberinfrastructure.<br /><br />Assembly is just one part of the big picture!</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: U Mass - C-reactive Peptide
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of C-reactive peptide reflect systemic inflammation and may be altered in obesity.</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 C-reactive Peptide test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Use of systemic antibiotics in patients with COVID-19 in Colombia. A cross-sectional study
AUTHORS: Jorge        Machado Alba

[DESCRIPTION]
Abstract 

Antibiotics are frequently prescribed to patients with COVID-19. The aim was to determine the pattern of use of systemic antibiotics in a group of patients diagnosed with COVID-19 in Colombia in the 2020-2022 period. This was a descriptive cross-sectional study designed to identify antibiotics prescription patterns for patients diagnosed with COVID-19 treated in 8 clinics in Colombia. The AWaRe tool of the World Health Organization (WHO) was used to classify the antibiotics. A total of 10,916 patients from 181 cities were included. The median age of the patients was 57 years, and 56.4% were men. A total of 29.7% of the patients required intensive care unit (ICU) care, and 20.2% required invasive mechanical ventilation. A total of 57.5% received antibiotics, especially ampicillin/sulbactam (58.8%) and clarithromycin (47.9%). Most of the antibiotics were classified as Watch (65.1%), followed by Access (32.6%) and Reserve (2.4%). Men (OR:1.29; 95% CI:1.17-1.43), older adults (OR:1.67; 95%CI:1.48-1.88), patients with dyspnea at admission (OR:1.26; 95%CI:1.13-1.41), rheumatoid arthritis (OR:1.94; 95%CI:1.17-3.20), and high blood pressure (OR:1.45; 95%CI: 1.29-1.63), patients treated in-hospital (OR:5.15; 95%CI: 4.59-5.77), patients admitted to the ICU (OR:10.48; 95%CI:8.82-12.45), patients treated with systemic glucocorticoids (OR:3.60; 95%CI:3.21-4.03) and vasopressors (OR:2.10; 95% CI: 1.60-2.75), and patients who received invasive mechanical ventilation (OR: 2.37; 95% CI: 1.82-3.09) were more likely to receive a systemic antibiotic.Most of the patients diagnosed with COVID-19 received antibiotics despite evidence showing that bacterial coinfection is rare. Antibiotics from the Watch group predominated, a practice that goes against WHO recommendations.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
How to run uproc v1.2.0 (Meinicke, 2014) through the iMicrobe plaform.
 
The ultrafast protein classification (UProC) toolbox implements a mosaic matching algorithm for large-scale protein annotation analysis. UProC is up to three orders of magnitude faster than profile-based methods and achieved higher sensitivity on unassembled short reads (100 bp) from simulated metagenomes. UProC does not depend on a multiple alignment of family-specific sequences. Therefore, in addition to the protein domain classification according to the Pfam database, UProC also provide the detection of KEGG Orthologs.
 
More informations about centrifuge can be found here : http://uproc.gobics.de/

[BEFORE_START]
You need a working Cyverse account to connect to iMicrobe.
To explore how to log into iMicrobe, read the dedicated protocol. 
Your dataset of interest should be metagenomic reads, in a fasta or fastq format.


In iMicrobe, there is several ways to run an app on a dataset (from the cart, from your personnal datastore and form an URL). If you need more information on how to run an app, read the protocol associated.

[GUIDELINES]
More informations and details about Uproc can be found here : https://github.com/gobics/uproc
 

Several parameters are available to the user through the iMicrobe app. 

PROTEIN CLASSIFICATION OPTIONS:
-P N       --pthresh    N
    Protein threshold level. Allowed values:
        0   fixed threshold of 0.0
        2   less restrictive
        3   more restrictive
    Default is 3 .

-l         --long
    Use long read mode (default): Only accept certain ORFs (see -O below) and
    report all protein scores above the threshold (see -P above).

-s         --short
    Use short read mode: Accept all ORFs, report only maximum protein score
    (if above threshold).

-O N       --othresh    N
    ORF translation threshold level (only relevant in long read mode).
    Allowed values:
        0   accept all ORFs
        1   less restrictive
        2   more restrictive
    Default is 2.



Note than the iMicrobe app runs automatically the job with the following output format parameters, and therefore produces 6 output files (*.kegg|pfam28.preds, *.kegg|pfam28.stats, *.kegg|pfam28.counts) :

-p         --preds
    Print all classifications as CSV. The printed fields are in the following order :
        n: sequence number (starting from 1)
        h: sequence header up to the first whitespace
        l: sequence length (this is a lowercase L)
        F: ORF frame number (1-6)
        I: ORF index in the DNA sequence (starting from 1)
        L: ORF length
        f: predicted protein family
        s: classification score

-f         --stats
    Print "CLASSIFIED,UNCLASSIFIED,TOTAL" sequence counts.

-c         --counts
    Print "FAMILY,COUNT" where COUNT is the number of classifications for
    FAMILY



[STEPS]
SECTION: Run uproc on metagenomic reads
?.