eamag/protocols-with-steps huggingface dataset:
TITLE: RNA isolation and RT-qPCR for Dengue, Chikungunya and Zika Viruses
AUTHORS: Maria Fernanda Avila Mejia, Pei-Yun Shu, Dar-Der Ji

[DESCRIPTION]
This protocol is for dried blood spot (DBS) sample collection, the extraction of viral RNA from dried blood spots, and the following RT-qPCR setup for the detection of Dengue, Chikungunya, and Zika viruses.

[BEFORE_START]
During RT-qPCR preparation, it is important to avoid DNA cross-contamination. Use an area exclusively for RNA work.

Clean the working area and all equipment with 10% bleach. 

Dispose of contaminated sharps and waste appropriately 

[STEPS]
SECTION: RNA Viral Extraction
2. 1. Use the paper puncher to punch a 6-mm punch in the dried blood spot. Sterilize the paper puncher with fire and make a clean punch after each use to avoid cross-contamination.

2.  Extract Viral RNA utilizing the QIAamp Viral RNA Mini Kit according to the manufacturer's instructions..
SECTION: RT-qPCR
3. 3. Prepare the RT-qPCR master mix using the Quantinova SYBR Green RT-PCR Kit for the three arboviruses. Three final volumes of 25 µl of PCR mixes contained 5 µL of RNA extract with individual DENV, CHIKV, or ZIKV primer pairs at a concentration of 0.3 µM, 0.3 µM, and 0.4 µM, respectively. 

  
 Primers
  
 
Master Mix 
               Dengue   Chikungunya Zika     Master Mix µL Master Mix µL Master Mix µL   RNA Sample 5 RNA Sample 5 RNA Sample 5   ddH20 3.75 ddH20 4.5 ddH20 4   SYBR green 12.5 SYBR green 12.5 SYBR green 12.5   RT Mix 0.25 RT Mix 0.25 RT Mix 0.25   Primer-F (0.3 µM) 0.75 Primer-F (0.3 µM) 0.75 Primer-F (0.4 µM)) 1   Primer-R1 (0.3 µM) 0.75 Primer-R (0.3 µM) 0.75 Primer-R (0.4 µM) 1   Primer-R2 (0.3 µM) 0.75 Rox 1.25 Rox 1.25   Rox 1.25           Total 25   25   25  
 
4. Process the RT-qPCR in the Applied Biosystems StepOnePlus™ Real-Time PCR System with the following conditions:

 
    1.   RT 
5. A sample was considered positive if the fluorescence curve crossed the threshold line within 40 cycles (Cq<40); and if the melting curve for DENV was around 80.25°C for DENV-1, 81.75°C for DENV-2, 80.,5°C for DENV-3 and DENV-4 for 83°C, for CHIKV and ZIKV was between 81°C and 82°C. Send positive samples for sequencing.
SECTION: Blood Sample Collection
1. 1.  Wear gloves and label the filter paper to be used before collecting DBS.

2.  Clean the finger from which sample will be collected with  75% alcohol. 

3.  Fingerprick with a small lancet after alcohol dries off. Apply gentle pressure to the finger and allow a large drop of  to collect at the puncture site. 

4. Remember NOT to press the filter paper against the puncture wound, rather allow the blood to drop and saturate the full circle. Avoid smering the blood spot.

5.  After the blood spots are completely on the filter paper, allow it to dry before placing it in a sealed plastic bag.

6.  Store the  filter paper in the sealed plastic bag with a desiccant package and then store it in closed box. Avoid exposure to the sun.
TITLE: Slice Preparation with Tissue Dissociation - Mouse Protocol
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure for transcardial perfusion of an adult mouse, followed by brain dissection, embedding, slicing, blockface imaging, ROI microdissection, and tissue dissociation. The output of this protocol is a cell suspension that can be sorted via FACS for single-cell transcriptional profiling.</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: Multi-electrode array recordings
AUTHORS: Nicola Biagio Mercuri

[DESCRIPTION]
MEA recordings in acute slices

[STEPS]
SECTION: Spike detection
1. Individual slices are placed over an 8 × 8 array of planar microelectrodes, each 20μm×20μm in size,
with an interpolar distance of 100 μm (MED-P2105; Alpha MED Sciences, Kadoma, Japan). Slices are positioned over the multi-electrode array under visual control through an upright microscope (Leica DM-LFS, Leica Microsystems, Wetzlar, Germany) so that most of the planar electrodes are covered by the
SNc area. The location of the medial terminal nucleus of the accessory optic tract is taken as a reference for the SNc area.
SECTION: Spike detection
2. The slices are kept submerged in ACSF with a nylon mesh glued to a platinum ring. Particular care is required to maintain a continuous flow of warm (34◦C) oxygenated ACSF (6 ml/min), streaming right above the SNc area. This procedure allowed us to preserve the stable spontaneous firing of the neuronal population recorded.
SECTION: Spike detection
2.1. ACSF composition is (in mM):  126 NaCl, 2.5 KCl,1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose and 25 NaHCO3.. The solution is saturated with a mixture of O2/CO2 (95/5%).
SECTION: Spike detection
3. Voltage signals are acquired using the MED64 System (Alpha MED Sciences), digitized at 20 kHz and filtered (0.1–10 kHz) with a 6071E Data Acquisition Card (National Instruments, Austin, USA), using Conductor software (Alpha MED Sciences).
SECTION: Spike analysis
4. The fast transients corresponding to spontaneous action potentials are captured offline using Spike2 6.0 software (Cambridge Electronic Design Ltd, Cambridge, UK), using an amplitude threshold adjusted by visual inspection in each active channel.
SECTION: Spike analysis
5. Spikes may differ in shape and amplitude in each active channel, reflecting spontaneous action potentials arising from more than one neuron, therefore spike-sorting discrimination of multi-unit responses is often needed. This is achieved by generating spike templates with Spike2 6.0 software, sorted with a normal mixtures algorithm on independent clusters obtained from principal component data.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Can be used for diluting or resuspending phage preparations.

[GUIDELINES]
Materials needed:<br />NaCl (MW=58.44)<br />Tris-Cl (MW=157.64)<br />MgSO<sub>4</sub>-7H<sub>2</sub>O (MW=246.47)<br />Milli Q water<br /><br />This buffer can be prepared from powdered chemicals or from pre-made liquid stocks.&nbsp; It is easiest to prepare from liquid stocks.<br /><br />At the least, the final buffer should be 0.2um filtered, however, it is best if it is 0.02um filtered (using Anotop syringe filters) just prior to use.

[STEPS]
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TITLE: PONE-D-19-35784 Proximal renal tubular function in HIV-infected children on tenofovir disoproxil fumarate for treatment of HIV infection at two tertiary hospitals in Harare, Zimbabwe
AUTHORS: Runyararo Mano

[STEPS]
?. AbstractThis laboratory protocol describes urine and  blood sample collection and measuring the concentration of urine glucose and protein,  urine protein creatinine ratio, urine, and serum phosphate as well as urine and serum creatinine to determine the prevalence of proteinuria,  estimated glomerular filtration rate and proximal renal tubular function among HIV infected patients on tenofovir disoproxil fumarate.
?. 
?. Authors Runyararo Masingaidze-Mano [Department of Paediatrics, University of Zimbabwe College of Health Sciences], Mutsawashe F Bwakura -Dangarembizi,[Department of Paediatrics, University of Zimbabwe College of Health Sciencies], Charles C Maponga[School of Pharmacy, University of Zimbabwe College of Health Sciencies], Gene D Morse [Centre for Global Biomedical Sciences, University at Buffalo, New York, USA], Tsitsi G Monera -Penduka [ School of Pharmacy, University of Zimbabwe College of Health Sciences ], Takudzwa J Mtisi[Department of Clinical Pharmacology, University of Zimbabwe College of Health Sciences ], Tinashe Mudzviti [Newlands Clinic, Highlands, Harare, Zimbabwe], Hilda A Mujuru [ Department of Paediatrics, College of Health Sciences]
?. Keywords Proteinuria,  serum and urine phosphate, urine protein creatinine ratio, tubular function, glomerular filtration rate
?.
TITLE: Plasmid construction
AUTHORS: Minghao Chen, Xuefeng Ren

[DESCRIPTION]
plasmid construction

[STEPS]
1. Amplify the insert gene fragment by PCR with primers including 21 nt of overlapping sequence with the target gene.  
2. Linearize the backbone with restriction enzymes (NEB). 
3. Treat linearized backbone with quick CIP (NEB). 
4. Run  PCR products and linearized backbone in an agarose gel to confirm the size. 
5. Purify the DNA from gel using a Gel extraction kit (Bio Basic).
6. Ligate the linearized backbone and the insert with the T4 ligase (NEB). 
7. Transform the ligation products into home made competent cells. 
8. Perform colony PCR to screen for colons that with inserted gene. 
9. Sequence to verify that the inserted gene is correct.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-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 derived from</p>
<p>[Reference: Processing guidelines for permanent epoxy negative photoresist SU8 2025, SU8 2035, SU8 2050 and SU8 2075, MicroChem company<strong>]</strong></p>

[STEPS]
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TITLE: Yale - Blood Glucose
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to measure glucose concentrations. Glucose is measured by the enzymatically coupled reactions of hexokinase and glucose-6-P dehydrogenase. The rate of NADH formation is monitored by the change in absorbance at 340 nm.</div></div>

[STEPS]
?. Calibrate Cobas for Glucose analysis by running a multi analyte standard and two control serum.
?. Sample Handling as performed by the Cobas Mira Plus.    a) Pipette 3 µL of sample into cuvette.    b) Absorbance is measured at 340 nm.    c) Add 100 µL of Glucose liquid reagent.    d) Mixture is incubated at 37ºC for 10 minutes.    e) Absorbance is measured at 340 nm. Change in absorbance is calculated.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For <a href="https://www.protocols.io/view/Enumeration-of-virus-particles-in-aquatic-or-sedim-dpy5pv" target="_blank">Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy</a> protocol.

[STEPS]
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TITLE: Intravenous (tail vein) injection of AAV into rat
AUTHORS: Janet R Keast, Nicole Wiedmann, Peregrine B Osborne

[DESCRIPTION]
This protocol is used to administer adeno-associated virus (AAV) in male and female rats. This procedure is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care.

[GUIDELINES]
All containers that are in contact with virus waste need to be properly labeled as biohazard waste, and disinfected appropriately following institutional safety requirements.
Virus stock needs to be placed on ice during surgeries.
For virus transport make sure to follow institutional safety requirements, e.g., double-containers are required for safe transport of biohazards (e.g. Eppendorf tube placed into 50ml Falcon tube).

[STEPS]
SECTION: Preparation
1. Determine the dose of virus to administer per rat. Divide the dose (viral genomes (vg)) by the titre (vg/ml) to calculate the volume of virus needed to inject one rat.  

Viral dosage can be determined using:   

SECTION: Preparation
2. In a screw-cap vial, prepare the solution to be administered. Add virus, and dilute with sterile 0.9% saline to a total 150 µl/rat. Briefly vortex each vial for 1 - 2 s before use. Transport the virus on ice once it is ready for injection. 

SECTION: Tissue Harvesting
10. Anesthetise and fix animals by intra-cardiac perfusion, then remove tissues of interest for further study.
SECTION: Surgery
3. Anesthetise animal (2.5% isoflurane in oxygen, or as required for maintenance).
SECTION: Surgery
5. Place animal on heated pad.
SECTION: Surgery
6. Apply 70% Ethanol wipes to tail and turn 45º for a visible presentation of tail vein. If unable to visualise the vein, warm the area.
SECTION: Surgery
7. Introduce a U-100 insulin syringe into the tail vein. Withdraw the barrel slightly to confirm location in the vein. Gently inject the virus.
SECTION: Surgery
8. Apply light pressure over the injection site for approximately 30 s to prevent backflow.

SECTION: Surgery
9. Return rat to housing cage and monitor for any evidence of swelling or complications at the injection site. Continue to monitor over the period of incubation for adverse reactions.
SECTION: Surgery
4. While the rat is being induced, load a U-100 insulin syringe with virus. Remove the dead space in the syringe barrel by gently ejecting the virus back into the tube such that air bubbles are expelled. Load the syringe again and repeat the procedure until no bubbles remain in the barrel.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div style="left: 45.1137px; top: 176.402px; font-size: 15px; font-family: sans-serif; transform: scaleX(0.955321);">Transformation &amp; Storage Solution (2X TSS) enables researchers to take advantage of the simple system described by Chung et al. <sup>1</sup> for the preparation, long-term storage and transformation of competent E. coli. Early log-phase cells are suspended in 1X TSS: a solution containing polyethylene glycol, dimethyl sulfoxide, and divalent cations in a bacterial growth medium.</div>
<p> </p>

[STEPS]
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TITLE: JMN-MSMP Muscle bulk RNA seq
AUTHORS: ccherry

[DESCRIPTION]
RNA sequencing of muscle

[STEPS]
SECTION: Bulk RNA sequencing
1. cDNA synthesis and library preparation were performed using the TruSeq RNA Library Prep Kit V2 following the manufacturer's protocol. 
SECTION: Bulk RNA sequencing
2. The Agilent BioAnalyzer system was used to perform quality control on samples during the library prep process as suggested in the manufacturer's protocol and RNA samples with RIN scores below 7 post-cDNA synthesis were excluded from analysis.
SECTION: Bulk RNA sequencing
3. Libraries were pooled and sequenced to a target depth of 30M unique paired end 50bp x 50bp reads per sample using a NovaSeq 6000.
TITLE: RPA from banana DNA
AUTHORS: Théo Nass, Luc Gabel

[STEPS]
?. [Banana DNA extraction]
Put chopped banana in a closed ziploc-like bag
?. [Banana DNA extraction]
Add diluted 1:10 dish soap solution and 1g table salt to fruits
?. [Banana DNA extraction]
Mash it until homogeneous mixture is obtained
?. [Banana DNA extraction]
Strain the mixture through a household coffee filter
?. [Banana DNA extraction]
Add 25 ml isopropyl alcohol to strained liquid
?. [Banana DNA extraction]
Let it sit for 5 min until phase separation
?. [Banana DNA extraction]
Collect upper white layer (precipitated DNA) with a toothpick, place it on a new coffee filter and wash it with 70% ethanol
?. [Banana DNA extraction]
Pat it dry with paper towels
?. [Banana DNA extraction]
Dissolve DNA in 1 ml TE buffer
?. [Nanodrop]
Perform a NanoDrop analysis
?. [Storage]
Set 1 μl aside; store rest at 4°C
?. [Prepare amplification mix]
Prepare the rehydration solution for the RPA  ABC1experimentRPA control2Rehydration buffer5.9 μl5.9 μl310μM forward primer0.42 μl0.42 μl410μM reverse primer0.42 μl0.42 μl5280 mM magnesium acetate0.5 μl0.5 μl6Sample extract1 μl0 μl7Water1.76 μl2.76 μl8Total10 μl10 μl
ABC1experimentRPA control2Rehydration buffer5.9 μl5.9 μl310μM forward primer0.42 μl0.42 μl410μM reverse primer0.42 μl0.42 μl5280 mM magnesium acetate0.5 μl0.5 μl6Sample extract1 μl0 μl7Water1.76 μl2.76 μl8Total10 μl10 μl
?. [Prepare amplification mix]
Vortex and spin briefly
?. [Prepare amplification mix]
Rehydrate 1 RPA reaction pellet in each one of the rehydration solutions
?. [Prepare amplification mix]
Pipette up and down to resuspend pellet
?. [Prepare amplification mix]
Vortex and spin briefly
?. [RPA reaction]
Heat the reaction at 39°C for 20 minutes.
?. [Gel electrophoresis]
Run a gel electrophoresis:  ABCD1Lane1232SampleDNA ladderSamplePCR control
ABCD1Lane1232SampleDNA ladderSamplePCR control
?. [DNA purification step]
should do a standard DNA purif kit (which we don't have at the moment) so we'll try the same purif as we did for the banana DNA
TITLE: 10x Protocols: Visium v2 CytAssist FFPE Library Construction -- University of Minnesota TMCs (CG000495 Rev B)
AUTHORS: IOx Genomics, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Protocols from 10x Genomics for Visium Spatial Gene Expression v2 chemistry on FPPE samples with the CytAssist component.

Protocol ID# (CG) and Revision used: 
    10x protocol CG000495, Revision B -- Library Construction with CytAssist

Note: These protocols may not be the current version offered by 10x Genomics but were used to produce the specific datasets connected to them. Please review the 10x Genomics support website for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
1. 10x protocol CG000 495, Revision B (Library construction with CytAssist)
  

2. Additional Protocols/Resources
https://www.10xgenomics.com/support/spatial-gene-expression-ffpe
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<p>This protocol was designed and developed at this laboratory by Greg A. Smith and colleagues for Public Helath Virology, Queensland's Department of Health, prior to his departure. It has not been previously published.</p>
<p>The protocol aims explicitly to amplify DENV-2 viruses and not other dengue viruses. The assay targets the membrane glycoprotein precursor region and is designed as a qualitative test for investigating suspected human cases of DENV-2 infection.</p>
<p>This assay has been superseded by the <a href="https://www.protocols.io/view/dengue-virus-type-2-denv-2-mgb-taqman-denv2-2016mg-n7kdhkw" target="_blank" rel="noopener noreferrer"><span class="">Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assa</span>y.</a></p>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol and with PCR in general.</li>
</ul>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[GUIDELINES]
<div>
<p>This was a past assay that we no longer in use.</p>
<p>For the best DENV-2 TaqMan assay, please refer to our recommended protocol:</p>
<p>Dengue virus type 2 (DENV-2) MGB TaqMan (DENV2-2016MGB) assay</p>
<p><a href="https://www.protocols.io/view/dengue-virus-type-2-denv-2-mgb-taqman-denv2-2016mg-n7kdhkw" target="_blank" rel="noopener noreferrer">https://www.protocols.io/view/dengue-virus-type-2-denv-2-mgb-taqman-denv2-2016mg-n7kdhkw </a></p>
</div>

[STEPS]
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TITLE: Quick estimation of molecular weight from gel images with an ImageJ macro, MolecularWeightEstimator
AUTHORS: Kenji Ohgane

[DESCRIPTION]
This protocol shares an ImageJ macro that enables easy and quick estimation of molecular weights from gel electrophoresis images, including the agarose gel electrophoresis for nucleic acids and the SDS-PAGE for proteins. By selecting the bands (size markers and samples of unknown size) and running this macro, you can estimate the size of your bands based on the curve fitted to your size marker data.

 

 

 


[STEPS]
SECTION: Installation of MolecularWeightEstimator macro into ImageJ
1. Download the macro file ("_MolecularWeightEstimator.ijm") anywhere in your local disk.  

SECTION: Installation of MolecularWeightEstimator macro into ImageJ
2. Start ImageJ.
SECTION: Installation of MolecularWeightEstimator macro into ImageJ
3. From the menu bar, select `Plugins > Install...`. Open the downloaded macro file, and save the macro in the `plugins` folder in the ImageJ directory.
SECTION: Installation of MolecularWeightEstimator macro into ImageJ
4. Restart ImageJ.
SECTION: Estimating MW from gel images.
5. Open an image file. 
 

SECTION: Estimating MW from gel images.
6. Start `ROI Manager', which can be found in the ImageJ menu, Analyze > Tools > ROI Manager.
 

SECTION: Estimating MW from gel images.
7. Select the rectangular tool, and select the bands of the size markers. To register each ROI into the ROI Manager, select a band and then click "Add" in the ROI Manager (or use a shortcut key like "Command+t" or "Ctrl + t").
 

SECTION: Estimating MW from gel images.
8. Also, register the bands with unknown molecular weight.
SECTION: Estimating MW from gel images.
9. Start the "Molecular Weight Estimator" plugin from the ImageJ menu (Plugins > MolecularWeightEstimator).
SECTION: Estimating MW from gel images.
10. Select the mode from either "vertical position" mode which uses the y-coordinate of the ROIs from the top of the image,  or "vertical length" mode which uses the vertical length of the ROIs as the electrophoretic mobility. Normally, the vertical position mode would be more easy and quick.
SECTION: Estimating MW from gel images.
11. Enter the molecular weights of your size markers. Use a comma to separate the values.
SECTION: Estimating MW from gel images.
12. Select the algorithm (function to fit).
 

SECTION: Estimating MW from gel images.
13. Click `OK` to run the macro to see the fitting result and a table showing the estimated molecular weights of your samples.