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

[DESCRIPTION]
<p>   ECMO inevitably increases the risk of infection over time; therefore, lowering infections acquired during ECMO should be further investigated and developed accordingly (Bizzarro, 2011). The degree of dysfunction by ECMO is evaluated using several methods, namely: ultrastructural changes in gut epithelium by TEM, plasma lipopolysaccharide (LPS) levels of bacterial products after receiving ECMO, etc. In particular, bacterial lipoteichoic acid (LTA) antigens in plasma can be measured over time by Western blot laboratory technique in association with the loss of gut barrier function below. The purpose of this pilot study is to evaluate how newly-developed ECMO reduces infection levels by controlling and stabilizing the overall hemodynamic status, based on the outcome of a Western blot.</p>
<p> </p>

[STEPS]
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TITLE: SW-3 SWAB STORAGE
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol describes swab storage.

[GUIDELINES]
OBJECTIVE

To outline steps for properly storing swab samples and nucleic acid samples purified from these samples.

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on storage of swab samples and their purified nucleic acid to preserve their integrity for downstream nucleic acid extraction and/or sequencing library preparation


MAINTENANCE OF EQUIPMENT

Decontaminate a PCR workstation by keeping the UV light on for 15 min.
 


[STEPS]
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
1. 
 
Precool 96-well microfuge tube racks on ice.

SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
2. Using preprinted adhesive labels or permanent markers, label 1.5 ml microfuge tubes.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
4. Once the rack is full or all swab samples have been completed, label the rack with a unique rack ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
5. Close the rack lid tightly, secure with clear Saran wrap and immediately transfer to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
6. Update the freezer inventory so samples can be tracked properly.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
7. 
 
In the clean PCR workstation, carefully transfer the eluted total nucleic acid to a 96-well PCR microplate, make sure to keep samples in the exact same locations corresponding to the rack where the original blood were stored.
 

SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
8. Cover the 96-well PCR microplate with adhesive film to prevent spill over or contamination.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
9. Label the film with a unique plate ID.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
10. Immediately transfer the 96-well PCR microplate to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
11. Update the freezer inventory so samples can be tracked properly.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
3. Transfer swab sample into the corresponding pre-labeled 1.5 mL microfuge tubes, cut and discard the swab shaft and keep the tip portion only, close the cap and put it onto the microfuge tube rack on ice sequentially.
TITLE: ATPase assay/ADP-glo kit
AUTHORS: Rania Abou El Asrar, Peter Vangheluwe

[DESCRIPTION]
This protocol is successfully used to assess activity of P5B-ATPases with polyamines as substrate. 

[STEPS]
1. Prepare master mix on ice: 5 µg microsomes, 2.5 µg DDM (Inalco, 1758-1350), reaction buffer (50 mM MOPS-KOH, pH 7, 100 mM KCl, 11 mM MgCl2 supplemented with 1 mM DTT and protease inhibitor (Sigmafast, Sigma, S8830-20TAB). 
2. Incubate master mix for 30 minutes at room temperature in overhead shaker. 
3. Pipet master mix (18.75 µl) into a white 96-well plate. 
4. Prepare serial dilution of the substrate to be tested in 0.1 M MOPS-KOH, pH 7.  
5. Pipet substrate dilution series (5 µl) into the 96-well plate.
6. Incubate for 5 minutes at room temperature then 5 minutes at 37 °C.
7. Add 5 mM pure ATP (1.25 µl) to initiate the reaction.
8. Incubate for 20 minutes at 37 °C followed by 10 minutes at room temperature.
9. Stop the reaction by addition of 25 µl ADP-glo reagent. 
10. Incubate at room temperature for 40 minutes.
11. Add 50 µl/well of ADP-glo max reagent. 
12. Incubate in the dark at room temperature for 60 minutes. 
13. Measure luminescence (integration: 500 ms)
TITLE: Wholemount Immunolabeling for GUT Samples
AUTHORS: Marthe Howard

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Wholemount Immunolabeling-Gut</div><div class = "text-block">This protocol is used for immunostaining large blocks of tissue.  The application that it was developed for is large pieces of mouse gut tissue.  Tissues shold be well washed prior to fixation.  </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Backgroud:</strong></p>
<p>Since sedation and analgesia are widely accepted by patients and even considered by many gastroenterologists as an integral component of the endoscopic examination, opioids are indispensible in managing pain. Nalbuphine is as effective as morphine as a perioperative analgesic but has not been compared directly with sufentanil in clinical trials.</p>
<p><strong>Objectives：</strong></p>
<p>The aims of this study were to compare the efficacy and safety of nalbuphine with that of sufentanil in patients undergoing colonoscopy and to determine the optimal doses of nalbuphine in this indication</p>
<p><strong>M</strong><strong>ethods</strong></p>
<p>Two hundred and forty consecutive patients aged 18–65 y with an American Society of Anesthesiologists classification of I–II and scheduled for colonoscopy were randomized to receive sufentanil 0.1 µg/kg (group S), nalbuphine 0.1 mg/kg (group N1), nalbuphine 0.15 mg/kg (group N2), or nalbuphine 0.2 mg/kg (group N3).  Baseline vital signs was recorded before the procedure. The four groups were monitored for propofol sedation using the bispectral index and pain relief was assessed using the Behavioral Pain Scale for non-intubated patients. The incidences of respiratory depression during endoscopy, nausea, vomiting, drowsiness, and abdominal distention were recorded in the post anesthesia care unit at 24 and 48 h after colonoscopy.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Here is a short explanation on how to add a work in progress protocol to a private group. This protocol shows how to add a protocol editable by multiple people to edit it <strong>before</strong> its final publication on protocols.io</p>
<p> </p>
<p>This is also an example protocol on how to explain to the members of the group on how to share protocols. </p>

[STEPS]
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TITLE: Protocol collection - &#34;Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism&#34;
AUTHORS: michela.deleidi, María José Pérez J., Pascale Baden, Federico   Bertoli, Hariam Raji

[DESCRIPTION]
Collection of protocols of Deleidi Lab used in the publication: "Glucocerebrosidase is imported into mitochondria and preserves complex I integrity and energy metabolism"

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
The Performance&nbsp;Index is calculated using the formula:<br /><br />PI=((#F5&times;5)+(#F4&times;4)+(#F3&times;3)+(#F2&times;2)+(#F1&times;1)+(#F0&times;0))/(#FT )<br /><br />where #Fn is the number of flies in the tube n (being 0 the initial tube and 5 the last tube), and&nbsp;#FT is the total number of flies. A higher index means a more positive response to light. In each experiment a PI is calculated for the wingless flies and other for the intact flies.<br /><br /><img src="https://s3.amazonaws.com/pr-journal/mec3kn.png" alt="" />

[STEPS]
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TITLE:        ELISA for quantification of transforming growth factor beta (TGF-β) in human serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Transforming growth factor beta (TGF-β) is a multifunctional cytokine belonging to the transforming growth factor superfamily. TGFB proteins are made by all white blood cell lineages.It is a multipotent growth factor affecting cell differentiation, tissue repair, cell proliferation, matrix production and apoptosis. [1]</div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Clark DA, Coker R. Transforming growth factor-beta (TGF-beta).</span><span style = "font-style:italic;">Int J Biochem Cell Biol</span><span>. 1998;30(3):293-298. doi:10.1016/s1357-2725(97)00128-3</span></div></div>

[STEPS]
?. An anti-humanTGF-β coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human TGF-β present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-TGF-β antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-TGF-β antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 20 min.
?. A colored product is formed in proportion to the quantity of TGF-β present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human TGF-β standard dilutions and the human TGF-β sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Initiating and monitoring natural infection of mice by bioluminescent Citrobacter rodentium 
AUTHORS: Hannah Read, Priyali Patel, Siouxsie Wiles

[DESCRIPTION]
Citrobacter rodentium is a Gram-negative bacterium that infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. We routinely use a bioluminescent derivative of C. rodentium called ICC180 so that we can monitor infection dynamics non-invasively using biophotonic imaging.

Experimental infection is typically initiated via oral gavage but we have previously shown that C. rodentium rapidly spreads between infected and uninfected animals and that bacteria shed from infected mice are 1000 times more infectious than laboratory-grown bacteria with a different tissue tropism.1,2 

In this collection of protocols, we describe the different methods for establishing infection by natural transmission and how to monitor infection dynamics using biophotonic imaging, viable counts, and luminometry.3  

1. Wiles, S., Dougan, G., & Frankel, G. (2005). Emergence of a 'hyperinfectious' bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract. Cellular microbiology, 7(8), 1163–1172. https://doi.org/10.1111/j.1462-5822.2005.00544.x
2. Bishop, A. L., Wiles, S., Dougan, G., & Frankel, G. (2007). Cell attachment properties and infectivity of host-adapted and environmentally adapted Citrobacter rodentium.Microbes and infection,9(11), 1316–1324. https://doi.org/10.1016/j.micinf.2007.06.006
3. Read, H. M., Mills, G., Johnson, S., Tsai, P., Dalton, J., Barquist, L., Print, C. G., Patrick, W. M., & Wiles, S. (2016). The in vitro and in vivo effects of constitutive light expression on a bioluminescent strain of the mouse enteropathogen Citrobacter rodentium. PeerJ, 4, e2130. https://doi.org/10.7717/peerj.2130

[STEPS]
TITLE: Sequencing at DNBSEQ-G400 platform with PE200 and SE400 modes
AUTHORS: Xiaohuan Sun, Yuehua  Hu, Zewei  Song

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for microbial short amplicon sequencing at DNBSEQ-G400 (previously known as BGISEQ-2000, RRID:SCR_017980) sequencing platform with PE200 and SE400 modes. DNBSEQ-G400 is a sequencer developed by MGI, using DNA nanoball and combinational probe anchor synthesis technoology.</div><div class = "text-block">Note:</div><div class = "text-block">This sequencing set is compatible with the libraries prepared by MGI Library Prep Kits. The size distribution of inserts should be between 400-600 bp.</div></div>

[STEPS]
?. Place the library on ice until use. Remove Make DNB Buffer, Low TE Buffer and Stop DNB Reaction Buffer from storage and thaw reagents at . Thaw Make DNB Enzyme Mix I for approximately  on ice. After thawing, mix reagents using a vortex mixer for . Centrifuge briefly and place on ice until use.
0 Room temperature
?. The DNBSEQ-G400RS sequencing flow cell contains 4 lanes. DNB can be loaded onto the flow cell using the sequencer, the MGIDL-200RS or the MGIDL-200H.
?. For general libraries, the ssDNA library concentration should be ≥ 2 fmol/μL and each Make DNB reaction requires 40 fmol library. The volume of each Make DNB reaction is  and the required library input for each Make DNB reaction is calculated as followed:ssDNA library input (μL) = 40 fmol / library concentration (fmol/μL)
100 µl
?. Mix ssDNA libraries, low TE buffer and Make DNB buffer as Make DNB reaction 1. Put Make DNB reaction 1 into a PCR machine and start the primer hybridization reaction with following steps:  for ,  for ,  for .
95 °C
65 °C
40 °C
?. Add  Make DNB Enzyme Mix I and  Make DNB Enzyme Mix II into Make DNB reaction 1 to form Make DNB reaction 2. Carry out Rolling circle amplification (RCA) for  at . After, add  DNBs stopping buffer to stop the RCA reaction.
40 µl
4 µl
30 °C
20 µl
?. Take  DNB, use Qubit® ssDNA Assay Kit and Qubit® Fluorometer to quantify the DNB. Sequencing requires a minimum DNB concentration of . If the concentration is lower than , make a new DNB preparation.
2 µl
?. Add  DNB load buffer II and  Make DNB Enzyme Mix II(LC) to  DNB product from the last step, and place it in the MGIDL-200H (the DNBs loading machine).
8 µl
0.25 µl
25 µl
?. Prepare the sequencing cartridge. Remove the Sequencing Reagent Cartridge and the High-throughput Sequencing Refill Kit (only for SE400 and PE200 sequencing) from storage. Thaw in a room temperature water bath until completely thawed. Store at 2-8°C storage until use (or thaw in 2-8°C fridge one day in advance). Invert the cartridge 3 times to mix before use. Shake the cartridge violently in all directions for 10-20 times until no visible layers can be seen in the cartridge.
?. Select sequencing modes PE200 or SE400. Sequencing is initiated after the sequencing reagent cartridge preloaded and sequencing chip was installed. When the whole sequencing was finished, the binary file with bases and quality score were converted into FASTQ format with Phred+33 quality score.
TITLE: Alpaca Nanobodies Rapid Ag test
AUTHORS: Anne Berking

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The "Rapid Ag test based on Alpaca Nanobodies" is a test developed with single domain antibodies. This type of antibody is capable of effectively recognize the Spike protein on the surface of SARS-CoV2 leading a signal that is 10 times stronger than conventional antibodies. </div><div class = "text-block">It takes only 8 minutes from sample to result. The procedures for using this test were designed to ensure an easy use and interpretation, making it a quick and simple test.</div></div>

[STEPS]
?. [Sample Collection]
Collect saliva in the sample collection tube.
?. [Sample Analyse]
Add Solution 1 containing the saliva to the "Detection column"
?. [Sample Analyse]
Connect the column to the vacuum manifold and let the full content pass through the "Detection columns"
?. [Sample Analyse]
Add 20 mL of Solution 2 "Washing Buffer".
?. [Sample Analyse]
Mix saliva sample with 10mL of Solution 1 “Binding Solution” and mix gently for 2 minutes.
?. [Analysis of the Results]
Add 100 µL of Solution 3 "Detection Reagent" and analyze after 2 minutes: blue color indicates antigen detection. Paper colour controls will be provided.
TITLE: Curva de Padrão Fosfato 16/11/20
AUTHORS: Mariana Jacques

[STEPS]
?. [Procedimento: ]
Em uma placa de  ___ poços colocar :50 μL de H2O MiliQ
?. [Procedimento: ]
A1: 50 μL Pi 40 μM( Realizar diluições seriadas)
?. [Procedimento: ]
Adicionar 25 μL MR em cada poço da placa
?. [Procedimento: ]
Preparar Revelador 4:1 Verde de Malaqueta: Molibidato de Amônioesperar 30' e filtrar
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Adicionar 200 μL de Revelador por poço
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Esperar 5-10 min ler no DO 660 nm
TITLE: VTEA - a tool for 3D tissue cytometry: 7-Click Analysis
AUTHORS: Seth Winfree, Daria Barwinska

[DESCRIPTION]
VTEA is a tool for 3D tissue cytometry (3DTC) of large 3D fluorescence light microscopy image volumes ranging from 50-100s of microns thick. The objective was to unify image processing, segmentation, data exploration and analysis in one package. 

VTEA is a tool that is…
…free and easy to get. It utilizes ImageJ/Fiji as the distribution platform. .
...easy to use. VTEA is designed to organize the most common workflow in 3DTC inclusive of image processing (to manage imaging artifacts), segmentation (extensible to bring in multiple approaches into a common framework of analysis) and exploration and analysis with flow cytometry like plots, gating, mapping to image with ROI gating and tools for high dimensionality data.
…compatible with mesoscale 3D analysis. VTEA is built to handle massive datasets and to operate seamlessly on an embedded database to enable analysis of 100’s of thousands of cells and will incorporate tools for clustering and dimensionality reduction.
…built around referencing the original image. The power of 3DTC in VTEA enables the localization of identified cells in the analysis space in the original image, in situ, and in 3D.

[BEFORE_START]
VTEA runs as a FIJI (ImageJ) plugin. 
Once VTEA is installed, the VTEA menu will appear on top of the FIJI menu strip. 

[STEPS]
1. Select the VTEA menu (Click #1) and then "VTEA 1.1.6" (Click #2). The version number will change with ongoing updates.
  

2. If no images are open in FIJI/ImageJ, use the open image button (Click #3). This will open up a standard FIJI open image file menu and automatically load an image into the VTEA workflow. 
  

3. If the image quality is sufficient for segmentation, preprocessing can be skipped by selecting "Process" button (Click #4). A separate sketch describes image processing and VTEA's functionality including combining image processing steps and previewing. 
  

4. The processed image is automatically moved to the segmentation setup after the image processing steps are completed. An image is also generated by the image processing steps that may be closed. The original image can also be closed at this time. Select the edit settings button (Click #5), to set and adjust the segmentation settings. 
  

5. Two windows are used to setup segementation in VTEA, the settings window and, if applicable, a threshold preview. Segementation can be previewed by drawing an ROI on the threshold preview and selecting the "eye" button. 
To continue close the setup window by clicking on the green checkmark button  (Click #6). To start the segmentation select the "Find Objects" button, (Click #7).
  

6. Once the segmentation is completed, VTEA's MicroExplorer window opens with a scatter plot of the processed data. 
In this scatter plot every dot represents a segmented cell. Where the dot is plotted is dependent upon the measured features plotted on the two axes and set by the drop-down menus for **X**, **Y** and **Color** respectively.
TITLE: ssDNA2.0: Adapter/splinter mix
AUTHORS: Matthias Meyer, Anna Schmidt, Sarah Nagel

[DESCRIPTION]
Protocol for the preparation of decontaminated (i.e., nuclease-treated) Adapter/splinter mix for the first adapter ligation in 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: No Title
AUTHORS: 

[DESCRIPTION]
Recent advances in single cell mRNA sequencing technology such as Drop-seq (Cell, 2015) have allowed the investigation of cellular composition and processes within complex tissues. Although some fresh tissue samples are available for prospective studies, there is an abundance of archived clinical samples readily available in biobanks to investigate different biological processes. However, archived tissues are not easily amenable to tissue digestion, yielding cells with compromised integrity. An alternate method to single-cell sequencing called DroNc-seq (Nature Methods, 2017) employs the use of single nuclei. DroNc-seq takes advantage of the higher structural integrity of the nuclear membrane as compared to the cellular membrane. This permits exploration of tissues for which a viable single-cell suspension cannot be obtained, such as cardiac tissue. With growing interest in cataloguing cardiac transcriptomic data for the Human Cell Atlas Project, the need for optimized nuclei isolation protocols proves critical. Here, we develop a method for obtaining single-nucleus suspensions from snap-frozen human heart tissue for use in DroNc-seq, expanding the variety of samples from which we can obtain transcriptomic data.

[BEFORE_START]
Heart tissue samples were collected in 1.5 mL microfuge tubes and snap-frozen before attempting nuclei isolation.

[GUIDELINES]
Keep samples on ice throughout procedure to prevent RNA degradation.

[STEPS]
SECTION: Reagent Prep
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SECTION: Nuclei Isolation
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SECTION: Tissue Prep
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Nuclei Isolation
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SECTION: Counting Nuclei
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SECTION: Diluting Nuclei for DroNc-seq
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