eamag/protocols-with-steps huggingface dataset:
TITLE: Extracorporeal membrane oxygenation meta-analysis of time-to-event data in respiratory failure in adults
AUTHORS: Hyunsuk Frank Roh, Chang-Guk Kim, Soon-Ho Chon, Jung Mogg Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purpose:</span><span>Time-to-event data of hazard ratios were used to generate a forest plot of mortality on extracorporeal membrane oxygenation (ECMO) in respiratory failure in adults.</span></div><div class = "text-block"><span style = "font-weight:bold;">Materials and Methods:</span><span>A systemic search was conducted in PubMed from 1975 to 2018. Among 4,121 articles, a total of 5 clinical reports comprising 1,014patients (333 of the patients who underwent ECMO and 681 of the patients who did not undergo ECMO) met the inclusion criteria.</span></div><div class = "text-block"><span style = "font-weight:bold;">Results:</span><span>The pooled hazard ratio of 0.64 (95% confidence interval (CI): 0.54, 0.76) suggests that the ECMO group comprising both veno-arterial (VA) type and a majority of veno-venous (VV) type was significantly associated with a reduction in mortality compared with the non-ECMO group in respiratory failure. The results of VV-ECMO alone also showed significantly improved patient survival of respiratory failure with the hazard ratio of 0.67 (0.56, 0.81).</span></div><div class = "text-block"><span style = "font-weight:bold;">Conclusions:</span><span>The results of the ECMO group with VV and VA types, or the VV type alone revealed a reduction in mortality for patients with respiratory failure based on time-to-event data.Future investigation of the efficacy of VA-ECMO alone in respiratory failure may be more enlightening.</span></div></div>

[STEPS]
?.
TITLE: Overall protocol for MicroPOTS LCMS top down proteomics of pancreas tissue sections
AUTHORS: Mowei Zhou, James M Fulcher, Yen-Chen Liao, Ljiljana.PasaTolic

[DESCRIPTION]
This is the overall workflow for LCMS top down proteomics of pancreas functional units from tissue sections using the MicroPOTS platform. The expected outcomes are proteoform identification and quantitation values from selected tissue functional units.

[STEPS]
SECTION: Tissue collection
1. The tissue sections were prepared and shipped from Vanderbilt-TMC following the protocol below:

 

SECTION: Sample preparation
2. Functional units (islet and acinar) were dissected and collected into the microPOTS platform using the method below:
 

SECTION: Data Acquisition
3. The samples were analyzed by LCMS top down proteomics as described below:
 

SECTION: Data Analysis
4. LCMS datasets were analyzed for proteoform identification and quantitation. The final results are reported.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Objective: To feed individual house flies a specific amount of bacteria in order to
determine bacteria “fate” (persistence, via enumeration; spatiotemporal location, via
microscopy) and house fly immune response (whole fly or tissue-specific, via
downstream mRNA or protein expression analyses).

  
 


[BEFORE_START]
Note: The bacteria culture conditions (e.g. media including antibiotics) will vary based on the
bacteria species and plasmid, if any.

[STEPS]
SECTION: Preparing House Flies for Bacterial Feeding
?. 
SECTION: Preparing House Flies for Bacterial Feeding
?. 
SECTION: Preparing House Flies for Bacterial Feeding
?. 
SECTION: Preparing House Flies for Bacterial Feeding
?. 
SECTION: Preparing bacterial culture
?. 
SECTION: Preparing bacterial culture
?. 
SECTION: Feeding House Flies on Culture Droplets
?. 
SECTION: Feeding House Flies on Culture Droplets
?. 
SECTION: Droplet enumeration
?.
TITLE: Biochemical Measures of Neuropathy - Genotyping
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/biv44kcn.jpg" /></div></div>

[STEPS]
?. [PCR Amplification:]
Prepare a Master Mix containing the following volumes of reagents:
?. [PCR Amplification:]
Into each 0.5mL thin-walled tube, pipette 23.0-μL of Master Mix.
?. [PCR Amplification:]
Pipet 2.0 μL of DNA template (200 ng/μL) into thin-walled tube. Mix gently, and spin down.
?. [PCR Amplification:]
Amplify using the following program:
?. [PCR Amplification:]
Verify PCR product by separation on 2% agarose.
?. [PCR Amplification:]
Store tubes at –20°C until needed. If plates will be used within a few days they may be stored at 4°C.
TITLE: Production of Crude AAV Virus Extract
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to produce crude preps of AAV of any serotype.</div><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: Test with subprotocols, version2
AUTHORS: Lenny Teytelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a test.</div></div>

[STEPS]
?. [Day 1: Sample Prep]
This section has instructions for sample processing and preparation.
?. [Day 2: Culturing]
valuesvalues * 215=A2*217=A3*219=A4*2
valuesvalues * 215=A2*217=A3*219=A4*2
?. [Day 2: Culturing]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Preparation of Vibrio natriegens cell stocks for long-term storage at -80C. Principle is to remove any accumulated metabolic waste from overnight culture and resuspend in 20% glycerol final.</p>

[BEFORE_START]
<p>prepare 60% glycerol stock. </p>
<p>prepare LB3 media (high salt media composed of LB added with 3% NaCl final).</p>

[STEPS]
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol to in vitro transcribe sgRNAs from oligos for S. pyogenes Cas9. </p>

[BEFORE_START]
<p><strong>10X transcription buffer</strong></p>
<ul>
<li>300mM Tris-Cl pH 8.1</li>
<li>250mM MgCl2</li>
<li>0.1% Triton X-100</li>
<li>20mM spermidine</li>
<li>100mM DTT</li>
</ul>
<div> </div>
<div><strong>2x Denaturing gel loading buffer</strong></div>
<ul>
<li>95% deionized formamide</li>
<li>20mM EDTA pH 8.0</li>
<li>Dye (I typically use Bromophenol blue, Xylene Cyanol and Orange G in this. None overlap with sgRNA)</li>
</ul>
<div> </div>
<div><strong>6x Agarose gel loading buffer</strong></div>
<ul>
<li>25% Ficoll</li>
<li>Enough orange G so that the loading dye looks brighter than Orange Crush soda (~0.1%).</li>
</ul>
<p> </p>
<p><strong>Gel extraction buffer</strong></p>
<ul>
<li>1mM EDTA</li>
<li>300mM NaOAc pH 5.2</li>
<li>0.5 w/v SDS</li>
</ul>

[GUIDELINES]
<p>The protocol workflow is as follows:</p>
<p> </p>
<p>1) Order oligos</p>
<p>2) Anneal oligo, extend &amp; amplify using PCR</p>
<p>3) Gel purify PCR products (optional)</p>
<p>4) Transcribe</p>
<p>5) Bead cleanup or gel purify RNA</p>
<p> </p>
<p>Note that there are two G's inserted at the 5' end of all sgRNAs generated using this protocol that are outside the protospacer. These do not affect cleavage biochemically and are trimmed inside cells if nucleofected as an RNP.</p>
<p> </p>
<p>It's recommended to start with a 100 µL transcription because the volumes are convenient and this is typically enough sgRNA. Scale up as needed.</p>
<p> </p>
<p>References: </p>
<p><a href="http://dx.doi.org/10.7554/eLife.04766" target="_blank" rel="noopener noreferrer">Lin &amp; Staahl, eLife 2014</a>.</p>
<p><a href="http://www.cshlpress.com/default.tpl?cart=145853182538485303&amp;fromlink=T&amp;linkaction=full&amp;linksortby=oop_title&amp;--eqSKUdatarq=725" target="_blank" rel="noopener noreferrer">RNA: A Laboratory Manual</a> (CSHL Press) Changelog: 1.01 - added references 1.1 - added bead cleanup</p>

[STEPS]
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TITLE: Tail suspension test to assess depression/anxiety behavior in parkinsonian mice
AUTHORS: natalia.lopezgonzalezdelrey

[DESCRIPTION]
The Tail Suspension Test is a mouse behavioral paradigm measuring behavioral despair or “depression-like” behavior and learned helplessness.

[GUIDELINES]
All mice should be carefully monitored for any adverse effects during the test and are quickly removed if they display signs of unusual distress (i.e. constant vocalization or damage to their tails). 

[STEPS]
SECTION: Tail Suspension Test
1. The mouse must be suspended using a piece of adhesive tape. The tape should be strong enough to prevent the mouse from falling and should not damage the skin of the tail. Tape length may vary with specific systems) and should only be applied to the very end of the tail (with 2-3 millimeters of tail remaining outside of the tape).
SECTION: Tail Suspension Test
2. Total duration: 3 days.
Total test duration per day: 6 min
SECTION: Protocol
3. The test lasts for 6 min and the immobility time is usually measured during the final 4 minutes as nearly all mice attempt to escape in the first 2 minutes, but immobility scores can be reported for the entire time of suspension.
SECTION: Protocol
5. If a mouse climbs up his/her tail, they will be gently guided back down with a probe by the investigator and the trial is continued. Mice that climb up their tails for approximately more than 20% of the total trial time should be removed from the analysis.
SECTION: Protocol
6. All mice should be carefully monitored for any adverse effects during the test and are quickly removed if they display signs of unusual distress (i.e. constant vocalization or damage to their tails). 
SECTION: Protocol
7. The chambers are cleaned with approved disinfectant at the start and end of the day and before and after each animal is tested.
SECTION: Analysis
8. The total amount of immobility time (defined as the time during which the animal is hanging passively and motionless) is measured for each animal, and considered as an index of “depression-like” behavior.
SECTION: Protocol
4. Video record for 6 min.
TITLE: ImageJ Fluorescence Image Composition
AUTHORS: Kenneth Schackart, Kattika Kaarj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details how to use ImageJ software to create a composite image from fluorescence images taken with a microscope.</div></div>

[STEPS]
?. [Get ImageJ ]
Go to https://imagej.nih.gov/ij/download.html and download the software corresponding to your operating system.
?. [Get ImageJ ]
Sometimes ImageJ will be hard to find and not show up within your programs. You may have to manually go into your hard drive and open the software from where you downloaded it.
?. [Open images ]
Open your images that are to be superimposed in ImageJ. This can be done by either:Clicking File -> Open, and selecting the files.
?. [Convert images ]
Convert each image to 8-bit by clicking on the image, then going to Image -> Type -> 8-bit.Repeat this for each image.
?. [Merge images ]
Merge the images by selecting Image -> Color -> Merge Channels...
?. [Merge images ]
For the various channel colors, choose the image from the drop down menu. Typically, we would do:Red: TRITCGreen: FITCBlue: DAPIIn this way, each will resemble the original color image.
?. [Merge images ]
Press ok, and you will have a merged image.
?. [Convert images ]
Adjust the image to increase contrast, etc. An easy way to enhance the contrast without much effort is to select Process -> Enhance Contrast...
TITLE: Ki-67 Staining Protocol
AUTHORS: Sam Li

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

[STEPS]
?. Prepare 70% Ethanol and chill to -20°C.Tip: Do not freeze ethanol for long-term storage.
?. Prepare target cells of interest and wash 2X with PBS, centrifuging at 350xg for 5 minutes.
?. Discard supernatant and loosen the cell pellet by vortexing.
?. Add 3ml cold 70% ethanol drop by drop to the cell pellet while vortexing.
?. Continue vortexing for 30 seconds and then incubate at -20°C for 1 hour.
?. Wash 3X with BioLegend's Cell Staining Buffer (Cat. No. 420201) and then resuspend the cells at the concentration of 0.5-10 x 106/ml.
?. Mix 100µl cell suspension with proper fluorochrome-conjugated Ki-67 antibody and incubate at room temperature in the dark for 30 minutes.
?. Wash 2X with BioLegend's Cell Staining Buffer and then resuspend in 0.5ml cell staining buffer for fluorescence activated cell sorting (FACS), or flow cytometric analysis.Tip: Based on customer testing, Ki-67 staining is not recommended with our True-Nuclear™ Transcription Factor Buffer Set.
TITLE: Rockwerx Male Enhancement Reviews Where To Buy It?
AUTHORS: rockwerxmalenhancenteviews  

[DESCRIPTION]
Rockwerx Male Enhancement Reviews Where To Buy It?


[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes an ELISA assay  for measurement of Granzyme B (CTLA-1) in supernatants collected from T cell dependent cytotoxicity (TDCC) cultures after BiTE® treatment.</p>

[STEPS]
?. 
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TITLE: Injection of Viral Tracers by Nanoject
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the delivery of a neuronal tracer using the Nanoject II. The surgery uses a stereotaxic system to target specific brain coordinates in the mouse. </div><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: Water Extractable Organic Matter (WEOM)
AUTHORS: maggie.bowman

[DESCRIPTION]
The method covers the extraction of water extractable organic matter for soil organic matter characterization using FT-ICR-MS, LCMS, and TOC/TN. 

[STEPS]
SECTION: Extraction
1. Weigh out 6 g of air-dried soil in triplicate into a labeled 50 mL falcon tube

SECTION: Sample Filtering (LC-MS and TOC/TN)n
9. Thaw ~20 mLsample prior to filtering

SECTION: Extraction
2. Add 30 mLof Ultrapure DI (maintaining a 2 g to 10 mL ratio) using large pipette

SECTION: Extraction
3. Place samples onto orbital shaker for  

SECTION: Extraction
4. Once completed centrifuge samples at 6000 rpm, 8 min

SECTION: Extraction
4.1. If samples are still cloudy, centrifuge a second time
SECTION: Extraction
5. Transfer the supernatant to two centrifuge tubes and do not discard the soil pellet
SECTION: Extraction
5.1. 5 mL in a 15 mL centrifuge tube for FT-ICR-MS

SECTION: Extraction
5.2. ~20 mLin a 50 mL centrifuge tube for TOC/TN and LC-MS

SECTION: Extraction
6. Acidify the FT-ICR-MS subsample using2 µL of concentrated phosphoric acid (37%)

SECTION: Extraction
7. Freeze the TOC/TN and LC-MS sample on the side at -20 °C until filtration

SECTION: Extraction
8. Freeze FT-ICR-MS samples on their side in the -80 °C until SPE 

SECTION: Extraction
8.1. SPE Protocol:Gilson Automated SPE Protocol
SECTION: Sample Filtering (LC-MS and TOC/TN)n
10. Using a new clean syringe for each sample, pull up 50 mL up Ultrapure DI water

SECTION: Sample Filtering (LC-MS and TOC/TN)n
11. Attach a new 0.45 um filter to the syringe and press the water through the filter to rinse the filter.
SECTION: Sample Filtering (LC-MS and TOC/TN)n
12. Remove the filter without touching the tip and pull up the ~20 mLof thawed sample

SECTION: Sample Filtering (LC-MS and TOC/TN)n
13. Re attach the rinsed filter and discard the first 1 - 2 mLof sample filtered

SECTION: Sample Filtering (LC-MS and TOC/TN)n
14. Filter the sample into two falcon tubes, two 15 mL tube labeled for TOC and LC-MS respectively.

SECTION: Sample Filtering (LC-MS and TOC/TN)n
15. Filter 10 mL of solution into the LC-MS tube and all remaining sample into the TOC/TN tube. 

SECTION: Sample Filtering (LC-MS and TOC/TN)n
16. If the filter becomes difficult to press sample through, rinse a new filter using a clean (DI ONLY syringe using steps 3 and 4) and attach the rinsed filter to the syringe with sample. Discard the first 1 - 2 mL and continue filtering into the respective tubes. 

SECTION: Sample Filtering (LC-MS and TOC/TN)n
17. Store filtered samples in the freezer prior to analyses.
TITLE: XPRIZE SHINE - In-tube Fluorescent SARS-CoV-2 NP Test
AUTHORS: Jon Arizti-Sanz, Catherine A. Freije, Chloe K. Boehm, Sameed M. Siddiqui, Allen M. Goodman, Tinna-Solveig F. Kosoko-Thoroddsen, A'Doriann Y. Bradley, Jeremy Johnson, Pardis C. Sabeti, Cameron Myhrvold

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to perform a SHINE in-tube fluorescent assay to detect SARS-CoV-2 RNA from a self-collected nasopharyngeal sample. This protocol is intended for point-of-care use. All enzymatic components are provided as a single-test freeze-dried pellet for shelf-stable storage, and all steps of the protocol are performed at ambient temperature. The protocol requires a transilluminator or another equivalent blue light emitting device. The protocol presented here is an improved version of the method presented in Arizti-Sanz J*, Freije CA*, </span><span style = "font-style:italic;">et al.</span><span> Integrated sample inactivation, amplification, and Cas13-based detection of SARS-CoV-2. </span><span style = "font-style:italic;">bioRxiv</span><span> (2020). </span></div></div>

[STEPS]
?. [Sample Collection and Viral Lysis]
Open the nasopharyngeal (NP) collection tube and rotate the nasal swab (attached to the NP collection tube cap) 4 times around the inside of each nostril. Return the swab to the collection tube and cap the tube.    Nasopharyngeal collection tube contains necessary volume of FastAmp® Viral and Cell Solution.
?. [Sample Collection and Viral Lysis]
Mix NP sample and FastAmp® Viral and Cell Solution by vortexing the closed sample collection tube for .
?. [Sample Collection and Viral Lysis]
Wait , incubating sample at , before proceeding to Step 4.
0 Room temperature
?. [SARS-CoV-2 Detection]
Pipette  of Reagent Mix A into a single uncapped well of the 96-well plate containing lyophilized Reagent Mix B. Mix by pipetting up and down gently.
15 µl
?. [SARS-CoV-2 Detection]
Add  sample-viral lysis mix to Reagent Mix A and B well. Mix by pipetting up and down gently. Recap sample.
5 µl
?. [SARS-CoV-2 Detection]
Wait  , incubating sample at , before proceeding to Step 7.
0 Room temperature
?. [In-tube Fluorescent Readout and Automated Analysis]
Visualize the fluorescence of the sample using a transilluminator or equivalent blue light emitting device.
?. [In-tube Fluorescent Readout and Automated Analysis]
With the user-provided smart device such as a smartphone, open the HandLens application and select in-tube as the test type.
?. [In-tube Fluorescent Readout and Automated Analysis]
Take a photo of the plate, and select upload. The result of the test will appear on the smart device screen.
TITLE: SPARC C2 Spinal Cord Hemisection Protocol in Rats
AUTHORS: Elisa Gonzalez-Rothi, Marissa Ciesla, Latoya Allen, Gordon Mitchell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure for performing a left lateral C2 spinal cord hemisection in rats</div></div>

[STEPS]
?. Rats are anesthetized in a closed chamber (3.5% isoflurane in 100% O2) and transferred to a heated surgical station where anesthesia is maintained via nose cone at 2-2.5% in 100% O2 for the duration of the surgical procedure. Prep injection site for injection using aseptic methods approved by Institutional approval body (IACUC)
?. A 1 inch dorsal incision is made from the base of the skull to ~ the 6th cervical segment and the dorsal cervical muscles are dissected in layers to expose the C2 lamina.
?. A C2 laminectomy is performed to expose the dorsal C2 spinal cord.
?. The C2 and C3 dorsal roots are visualized and a durotomy performed just caudal to the C2 nerve roots.
?. A complete lateral hemisection is performed on the left side of the spinal cord, extending from the spinal midline to the lateral edge of the spinal cord using a microscalpel.
?. Using gentle aspiration, the left C2 hemisegment is removed (~1mm) between the C2 and C3 nerve roots, enabling visualization and consistency of lesion completeness.
?. Closure of Surgical Site:
?. The dura is sutured with 9-0 ethilon nylon suture.
?. The overlying muscles are sutured with 4-0 Polysorb suture.
?. The skin is closed with 9mm stainless steel surgical wound clips.
?. Post-surgical care:
?. analgesic administration (buprenorphine, 0.03 mg/kg, s.q. every 12 hours for 72 hours post-surgery).
?. anti-inflammatory administration (meloxicam, 1 mg/kg., s.q. every 24 hours for 48 hours post-surgery).
?. administration of Lactated Ringers Solution (5 ml, 2x/day, s.q.) and Diet Gel Boost (1-2mL, 2x/day, p.o.) until adequate volitional drinking and eating resume.
?. manual bladder expression (2x/day) until voluntary bladder voiding resumes.