eamag/protocols-with-steps huggingface dataset:
TITLE: Phosphorus Extraction - Bray Method
AUTHORS: maggie.bowman

[DESCRIPTION]
The method estimates the relative bioavailability of inorganic ortho-phosphate (PO4-P) in soils with acid to neutral pH. 

[STEPS]
1. Print the required labels needed for Bray extraction for each site, depth, and replicate. 
2. Weigh out 2 gof soil into labeled 50 mL centrifuge tubes.

3. Add 14 mL of Bray Extract solution (in cabinet under hood in 1521) using the pipet and electronic pipettor. 

4. Cap the samples and shake to mix. Add samples to foam block on shaker. 
1.1. 1000S_SITE_P_BRAY_DEPTH_REPLICATE
1.2. 1000S_SITE_Pext_BRAY_DEPTH_REPLICATE
2.1. For example: 1000S_PRS1_P_Bray_TOP_1
5. Shake samples at  .

6. Centrifuge samples (using centrifuge on west side of 1521) for4000 rpm, 8 min. 

7. Pour off liquid into a clean and labelled 15 mL centrifuge tube.

7.1. For example: 1000S_PRS1_Pext_Bray_TOP_1
8. Freeze samples on their side to prevent tube breaking in the -20 °C freezer (Staging Freezer 2) bottom shelf if unable to filter immediately.

9. Follow sample filtering protocol below when ready to filter extracts.
TITLE: Dual-target detection of Mpox virus
AUTHORS: Alan O'Dwyer, Tracy Lee, Michael Chan, Branco Cheung, Frankie Tsang, John Tyson, Kathleen Kolehmainen, Natalie Prystajecky, Agatha Jassem

[DESCRIPTION]
This procedure provides instructions for detecting Mpox virus and human beta-globin using a laboratory developed fast-enabled PCR assay on a Quantstudio 7 real-time PCR instrument.

To address the 2022 Mpox virus (MPXV) outbreak a real-time PCR targeting TNF receptor gene (G2RG) as described in the Journal of Virological Methods 2010 publication by Li et al. (1) was assessed and multiplexed with human betaglobin as endogenous control. 

In September 2022, the CDC released an alert on its Laboratory Outreach Communication System stating that three cases of Mpox were detected in California with a significant deletion in the TNF receptor gene. These cases were undetectable by the G2RG TNF-targeted qPCR (2). In response, a secondary MPXV target was required to multiplex with the existing G2RG target in order to detect any future cases with a TNF receptor deletion. Several targets were trialed and the MPXV GP113 gene was selected. 

This validation report presents the data supporting the use of a G2RG/GP113/HBG multiplex qPCR for the detection of Mpox virus.

[BEFORE_START]
Prepare a 20x oligo mix per the recipe in the materials section 

[GUIDELINES]
This procedure describes testing of extracted DNA. Sample extraction is not included.
Prepare PCR master mixes in a Clean Room. 
Perform all manipulations of samples and DNA in a genomic level PCR laboratory

[STEPS]
SECTION: Prepare master mix
1. Prepare the master mix cocktail according to the following table. Invert mixture gently several times to mix and quickly spin down.


 


SECTION: Mpox PCR
2. In a Biological Safety Cabinet, transfer 5µL of sample extract and controls to the optical plate in order:
Patient Sample Extracts
Positive and Negative Extraction Controls
MPXV Positive PCR control (Amplirun Monkeypox Virus DNA Control)
Matrix (BG) control
No Template Control (NTC)

Seal the plate with an optical plate film 

SECTION: Prepare master mix
1.1. Aliquot 15µL of master mix to each well of a MicroAmp Fast Optical 96 well reaction plate (0.1mL). Transfer plate to the PCR Genomic Room 
SECTION: Mpox PCR
2.1. Load the plate onto an ABI QuantStudio 7 Real-Time PCR instrument. Refer to Table 2 for fluorophore information. Refer to Table 3 for thermocycling conditions. Refer to Table 4 for recommended manual target threshold settings.


 



 



 


SECTION: Mpox PCR
2.2. Run the PCR and analyze results.
TITLE: MojoSort™ Human CD56 Nanobeads Protocol
AUTHORS: Ken Lau

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">"MojoSort™ Human CD56 Nanobeads Protocol</span><span></span></div></div>

[STEPS]
?. Prepare cells from your tissue of interest or blood without lysing erythrocytes.
?. In the final wash of your sample preparation, resuspend the cells in MojoSort™ Buffer by adding up to 4 mL in a 5 mL (12 x 75 mm) polypropylene tube.Note: Keep MojoSort™ Buffer on ice throughout the procedure.
?. Filter the cells with a 70 µm cell strainer, centrifuge at 300xg for 5 minutes, and resuspend in an appropriate volume of MojoSort™ Buffer. Count and adjust the cell concentration to 1 x 108cells/mL.
?. Aliquot 100 µL of cell suspension (107cells) into a new tube. Add 20µL of True-Stain Monocyte Blocker™, mix well and incubate at room temperature for 10 minutes. Scale up the volume accordingly if separating more cells. For example, if the volume of True-Stain Monocyte Blocker™ for 1x107cells is 20µL, add 200µL for 1 x 108cells. When working with less than 107cells, use indicated volumes for 107cells.
?. Resuspend the beads by vortexing, maximum speed, 5 touches. Add 10µL of Human CD56 Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, add 100 µL of Nanobeads for separating 1 x 108cells in 1 mL of MojoSort™ Buffer. When working with less than 107cells, use indicated volumes for 107cells.
?. Wash the cells by adding MojoSort™ Buffer up to 4mL. Centrifuge the cells at 300xg for 5 minutes.
?. Discard the supernatant.
?. Add 2.5mL of MojoSort™ Buffer.Note: If you observe aggregates, filter the suspension. To maximize yield, you can disrupt the aggregates by pipetting the solution up and down.
?. Place the tube in the magnet for 5 minutes.Optional: Take a small aliquot before placing the tube in the magnet to monitor purity and yield. Keep unused cells to be used as control or other applications if needed.
?. Pour out the unlabeled fraction. If these are your cells of interest, DO NOT DISCARD. Resuspend the labeled cells in 2.5 mL MojoSort™ Buffer.
?. Repeat steps 9-10 on the labeled fraction twice more for a total of 3 separations. Pool the unlabeled fractions and keep the labeled cells. The fraction that is not of interest may be useful as staining controls, to monitor purity/yield, or other purposes.Optional: Take a small aliquot to monitor purity and yield.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Lentiviral vectors pseudotyped with influenza surface glycoproteins represent an alternative to wild type virus for serological assays. The influenza neuraminidase (NA) has the ability to bud and release new virions with or without the contribution of Haemagglutinin (HA). Influenza pseudotypes expressing NA alone, or with HA, can be produced to evaluate the antibody response against NA using an enzyme-linked lectin assay (ELLA). The expression of an avian HA with human NAs has enabled the detection of specific antibody reponses against the human circulating subtypes of NA. This protocol describes the efficient production of NA pseudotypes for these ELLA assays. The pseudotypes have H11 HA on the surface as this will not react with human sera.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/2252nq6.jpg" data-src="https://s3.amazonaws.com/pr-journal/2252nq6.jpg" data-ofn="PV.jpg" /></p>
<p> </p>
<p>Figure 1. Cartoon schematic of lentiviral vectors pseudotyped with NA or HA+NA for use in ELLA assays.</p>

[GUIDELINES]
<p>phCMV1-H11 (A/duck/Memphis/546/1974 (H11N9)) was kindly provided by Dr Davide Corti, Institute for Research in Biomedicine, Switzerland. N1/Cal09 and N2/Tex12 were synthesised (WT sequence) and subcloned into plasmid expression vector pI.18 (Cox et al. 2002) by Genscript (U.S.A). HIV-1 derived packaging plasmid pCMVΔR8.91 (p8.91) was obtained from Dr Yasu Takeushi, University College London, originating from the laboratory of Dr Didier Trono (Naldini et al. 1996; Zufferey et al. 1997). Plasmid pCSGW originated from (Demaison et al. 2002), the GFP gene was replaced by that encoding firefly luciferase, forming the plasmid pCSFLW by Novartis Vaccines, Italy.</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Perturb-FISHv3
AUTHORS: loic binan, Sami Farhi, Brian Cleary

[DESCRIPTION]
This is the 07-21-2024 update version to this protocol protocol describes how the Sami Farhi lab, https://www.broadinstitute.org/bios/sami-farhi, carries out Perturb-FISH and was provided by Loïc Binan, https://sites.broadinstitute.org/opp/people/lo%C3%AFc-binan. See the author's paper for a wider context of this protocol: https://doi.org/10.1101/2023.11.30.569494 


Note: This protocol is being connected to part of the NIMH-initiated consortium SSPsyGene: https://sspsygene.ucsc.edu/, SSPsyGene has the aim to functionally characterize the contribution of multiple genes to neurodevelopmental and psychiatric disorders (NPD) resulting in a comprehensive phenotypic catalog.

[STEPS]
TITLE: Crude Subcellular fractionation of FAM177A1-GFP expressing cells
AUTHORS: Berrak Ugur, Michael G. Hanna, Pietro De Camilli

[DESCRIPTION]
This protocol details the crude subcellular fractionation of FAM177A1-GFP expressing cells.

[STEPS]
SECTION: Crude Subcellular fractionation
1. 
 

Culture and transfect HeLa cells as described in dx.doi.org/10.17504/protocols.io.eq2lyp55mlx9/v1 

SECTION: Crude Subcellular fractionation
2. 1440 min-2880 min after transfection, wash cells in 6 well plates with PBS.

SECTION: Crude Subcellular fractionation
2.1. Add 200 µL PBS (or fractionation buffer) to each well.

SECTION: Crude Subcellular fractionation
2.2. Scrape cells to release from well and transfer to a 1.7 mL eppendorph tube with additional 100 µLfractionation buffer wash. 

SECTION: Crude Subcellular fractionation
3. Spin the lysate at 1500 rpm, 5 min in a benchtop centrifuge.

SECTION: Crude Subcellular fractionation
4. Remove the supernatant and resuspend cell pellet in 1 mL of cold fractionation buffer.

SECTION: Crude Subcellular fractionation
5. Homogenize resuspended cells with cell cracker (Isobiotec; 8-12 strokes), 1 mL at a time.

SECTION: Crude Subcellular fractionation
5.1. Wash with 1 mL fractionation buffer between samples.

SECTION: Crude Subcellular fractionation
5.2. Always use new syringes for each sample.
SECTION: Crude Subcellular fractionation
6. Ultracentrifuge lysates in a Beckman-Coulter table-top ultracentrifuge (TLA100 rotor) at 50000 rpm, 60 min, 4 °C to pellet membrane.

SECTION: Crude Subcellular fractionation
7. After the centrifugation, transfer the supernatant to a new 1.7 mL Eppendorf tube and save it for western blot analysis.

SECTION: Crude Subcellular fractionation
8. Solubilize the membrane fractions from the bottom of the tubes using 4X Laemni buffer for western blot analysis.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is for comparing two different samples at the transcript level, using long reads that are mapped to transcripts.</p>
<p> </p>
<p><strong>Input(s)</strong>: stranded fastq files (see steps 1-8 of <a href="https://dx.doi.org/10.17504/protocols.io.n8ddhs6" target="_blank" rel="noopener noreferrer">Stranded Mapping from Long Reads</a>), transcript reference fasta file, annotation file</p>
<p> </p>
<p><strong>Output(s):</strong> transcript table, sorted by differential coverage, annotated with gene name / description / location</p>

[BEFORE_START]
<p>Obtain a transcript fasta file, and an annotation file. For the mouse genome, I use the following files:</p>
<p> </p>
<ol>
<li>Transcript [CDS] sequences from <a href="http://asia.ensembl.org/Mus_musculus/Info/Index" target="_blank" rel="noopener noreferrer">Ensembl</a>; <a href="ftp://ftp.ensembl.org/pub/release-92/fasta/mus_musculus/cds/Mus_musculus.GRCm38.cds.all.fa.gz" target="_blank" rel="noopener noreferrer">this file</a> is the most current at the time this protocol was created.</li>
<li>Annotation file obtained from <a href="http://asia.ensembl.org/biomart/martview" target="_blank" rel="noopener noreferrer">Ensembl BioMart</a> (Ensembl Genes -&gt; Mouse Genes) as a compressed TSV file with the following attribute columns:
<ul>
<li>
<div>Transcript stable ID</div>
</li>
<li>
<div>Gene description</div>
</li>
<li>
<div>Gene start (bp)</div>
</li>
<li>
<div>Gene end (bp)</div>
</li>
<li>
<div>Strand</div>
</li>
<li>
<div>Gene name</div>
</li>
<li>
<div>Chromosome/scaffold name</div>
</li>
</ul>
</li>
</ol>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Drug feeding protocol
AUTHORS: Mel Feany

[DESCRIPTION]
This protocol covers the steps for drug feeding in fly models.

[STEPS]
1. Collect flies for feeding:
-Clear bottles the day before collecting
-Select appropriate genotype for feeding
2. Prepare drugs to be fed:
-Thaw drugs
-Add 3 mL water to each vial
-Add appropriate number of ul drug to water, mix by swirling
3. Make fly food in vials that contain water and drug:
-Place the label that matches the drug you are using on the vial
-Add dry fly food to make a thick slurry, water will be absorbed in minutes
-Turn on gas and knock out flies. Put up to 40 flies into vial.
-Put the flies in a box in the 25 °C incubator

4. Flies are fed 3 doses of each drug. Flies are fed for a total of 10 days.
5. Harvest flies at 10 days and fix in formalin for histological analysis or process appropriately for other studies as needed.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We have developed a plaque assay for AaV with a modified protocol from Schroeder et. al 2002. </p>
<p> </p>
<p> </p>
<p><em>Protocol modified from</em>:</p>
<p> </p>
<p>Schroeder, D.C., et al.,<em> Coccolithovirus</em> (<em>Phycodnaviridae</em>): Characterisation of a new large dsDNA</p>
<p>algal virus that infects <em>Emiliania huxleyi</em>. Archives of Virology, 2002.147(9): p. 1685-1698.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: HPC Account Set up and Access
AUTHORS: Courtney Comrie

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol will provide instruction on where to access the HPC lab account, and where our data is stored.</div><div class = "text-block">Only for internal use.</div></div>

[STEPS]
?. Go to the main HPC information webpage, and select web interface under the Services section
?. [Access to HPC ]
Enter Dr. Hutchinson's email into the sponsor's email box so she can add you to the drive: hutchinsone@email.arizona.edu
?. After this is done you must wait till accepted onto the lab account.
?. [Access to HPC ]
Go to the main HPC information page, and select web interface under the Services section.
?. [Access to HPC ]
This should take you to a webpage with the option to "Manage Your Accounts," select this option.
?. [Access to HPC ]
Select HPC Account
?. [Access to HPC ]
Note: You only need to follow these steps if you have not logged onto the HPC web interface before. If you already have access then select web interface from step one, and enter your NetID and password when instructed.
?. [Lab directory]
Once access is granted, log into the web interface with your NetID.This will bring you to the page below.
?. [Lab directory]
Go to the Files tab and select /rsgrps (research groups)
?. [Lab directory]
/rsgrps displays all the research groups on the HPC directory. Select hutchinsone
?. [Lab directory]
You are now in our lab directory.
?. [Getting to HPC Desktop]
Return to On Demand Page.
?. [Getting to HPC Desktop]
Under the Interactive Apps tab select Ocelote Desktop.
?. [Getting to HPC Desktop]
Note: Never chose ElGato Desktop for our purposes.
?. [Getting to HPC Desktop]
.
?. [Getting to HPC Desktop]
For your settings on the Ocelote Desktop use the following guide:Run Time - Enter the amount of time you plan on clocking in that day i.e. use the run time as a tracker for your work.      Note: Be careful to give yourself enough time to accomplish jobs, or your session will end resulting in work      lost.     Core Count on a single node - Make this choice accourding to the amount of data you are processing. Large data files = more cores.Special Option - leave this blankPI Group - Enter HutchinsoneQueue - Leave as Standard      Note: Be careful to give yourself enough time to accomplish jobs, or your session will end resulting in work      lost.     Core Count on a single node - Make this choice accourding to the amount of data you are processing. Large data files = more cores.Special Option - leave this blankPI Group - Enter HutchinsoneQueue - Leave as Standard
?. [Getting to HPC Desktop]
Select Launch!
?. [Access to HPC ]
Log into your UA NetID.
?. [Access to HPC ]
Select the link on the page.
?. [Access to HPC ]
Select the notify sponsor option.
TITLE: TH Densitometry
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to quantify the density of dopaminergic projections to the striatum.

[STEPS]
1. Take 4 cryosections to equally sample the striatum and perform diaminobenzidine staining with a tyrosine hydroxylase antibody.
Note: I take every 12th section from Bregma 1.18mm to -0.22mm
2. Capture brightfield images of the dorsolateral striatum and convert to greyscale images.
3. Take 20 intensity measurements of the striatum and 5 intensity measurements of the corpus callosum (to account for background staining).
4. Average the corpus callosum intensities then subtract that from the striatal intensities then average the normalized striatal intensitied.
TITLE: Tube Radius Calculation
AUTHORS: Liv  Jensen

[DESCRIPTION]
This protocol details Tube Radius Calculation.

[STEPS]
SECTION: Fluorescence ratio calibration:
1. Measure the radius of the GUV and of the aspiration pipette from imaging data.
SECTION: Fluorescence ratio calibration:
2. Calculate radius from:

   

SECTION: Fluorescence ratio calibration:
3. In FIJI, create new image stacks from two rectangular selections: one containing a section of the membrane tube, and one containing the approximately horizontal section of the guv.
SECTION: Fluorescence ratio calibration:
4. Save these new stacks as .tif files and paste paths into segmentation and quantification script.
SECTION: Fluorescence ratio calibration:
5. Use the output of the segmentation and quantification script to calculate the ratio of fluorescence intensity of the membrane tube and the GUV surface (Itub / IGUV).

SECTION: Fluorescence ratio calibration:
6. Plot tube radius as calculated in step 2 vs. the fluorescence ratio calculated in step 5. Perform a linear fit of the data, recording the slope, Ktub, for use in subsequent experiments.

SECTION: Calculating tube radius with fluorescence images and Ktub:
7. In FIJI, create new image stacks from two rectangular selections: one containing a section of the membrane tube, and one containing the approximately horizontal section of the guv.
SECTION: Calculating tube radius with fluorescence images and Ktub:
8. Save these new stacks as .tif files and paste paths into segmentation and quantification script.
SECTION: Calculating tube radius with fluorescence images and Ktub:
9. Input Ktub as a parameter in the quantification function to normalize fluorescence ratio to tube radius.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Intended use:</strong></p>
<p>Kinetic system for Alanine Aminotransferase (ALT/ GPT) activity determination.</p>
<p> </p>
<p><strong>Teste Principle:</strong></p>
<p> </p>
<p><strong>ALT</strong> catalyzes the transfer of amino groups from Alanine to Ketoglutarate, yielding pyruvate and Glutamate. The pyruvate is reduced to lactate by for action of the lactate dehydrogenase (LDH) which oxidizes NADH to NAD<sup>+</sup>.</p>
<p>The reduction of the absorbance at 340 nm, as consequence of NADH oxidation, must be photometricaly monitored, and is proportional to the ALT activity in the sample.</p>
<p>Aminotransferase determination involves the following reactions:</p>
<p>L-alanine + Ketoglutarate ---(ALT) → Pyruvate + L-Glutamate</p>
<p>Pyruvate + NADH ---(LDH)→  NAD + L-lactate</p>
<p> </p>

[BEFORE_START]
<p>Spectrophotometer.</p>
<p>- Micropipettes and pipettes for measuring the stated volumes</p>
<p>- Water bath at temperature indicated under PROCEDURE.</p>
<p>- Stopwatch.</p>

[GUIDELINES]
<p>Reagents are for “in vitro” diagnostic use. Use the reagents according to the working procedures for clinical laboratories.</p>
<p>Storage and stability - Unopened reagents, when stored at indicated temperature, are stable up to expiration date shown on the label.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: 1.1 Bead beating
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Sample preparation for Western bolt</div></div>

[STEPS]
?. [Sample preparation for Western blot]
Bead beating
?. [Sample preparation for Western blot]
Bead size selection0.1 mm diameter for bacteria, 0.5 mm diameter for yeast, and 1 or 2.5 mm diameter for a chopped-up plant or animal tissue.Fill2 ml screw-cap microtube with0.5 gm of beads, keep the tubes in ice and add cells. Use screw-cap microtubes with integral o-ring seals in order to eliminate aerosol formation during the homogenization. Be sure there are no beads on the threads of the microtubes when screwing down the cap.
?. [Sample preparation for Western blot]
Operating the Mini-BeadBeater-8Insert one to eight microtubes into the chamber holder. Distribute symmetrically. Screw in the black plastic chamber cap, flat side down, until it is in contact with the tops of the microtube caps. Finally, screw on tight the white nylon wing nut.Set the toggle switch on the control box to 'Time' and select the speed. A typical setting for cell disruption is 3 minutes at Homogenize. If you are working with a heat-sensitive material, consider homogenizing for 1 minute, then cooling the vials in ice-water for 1 minute. Cycle for a total 'On' time of three minutes. Nucleic acid extraction does not need cooling.Start the homogenization by pushing the white button in the middle of the timer dial. The timer dial resets itself automatically at the end of the run..
CAUTION - While the MBB-8 is running, changing the Timer settings will damage the timer. If you must change the time setting, press, and hold down the white start button while changing the position of the timer dial.
?. [Sample preparation for Western blot]
Safety ConcernsOperate the MBB-8 with the black plastic hood over the chamber. This prevents the user from coming in contact with the shaker during operation and will help trap anything that might break free.The noise level of the disrupter exceeds 85dB. You may want to isolate the MBB-8 to a side room.  Do not isolate the MBB-8 in a cold room.  It will lead to premature motor failure and does not help to keep samples cold during homogenization.Centrifuge at 10,000 g for 5 minutes. Transfer the supernatant to an Eppendorf tube and spin again (10,000g for 5 minutes). Transfer the supernatant to a polypropylene microfuge tube(which is your total protein).
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<p><strong>Aseptic Removal of Femurs</strong></p>
<p> </p>
<p>Small beaker of 70% EtOH</p>
<p>Scissors</p>
<p>Dissection blades</p>
<p>Tweezers</p>
<p>Ice</p>
<p>50 mL Falcon with 25 mL cold phenol red free media or PBS on ice for each strain used.</p>
<p>Dissection board and pins (3)</p>
<p> </p>
<p><strong>Under tissue culture hood</strong></p>
<p> </p>
<p>3ml syringe</p>
<p>20G/ 26 G needle</p>
<p>*RPMI media (RPMI 10%FBS, 1% pen/strep, glutamine 2mM)</p>
<p>*RPMI 10%FBS <strong>without </strong>Pen/Strep – <em>PHENOL FREE FOR LDH</em></p>
<p>*Prepare day before</p>
<p>6 well plates</p>
<p>50ml tubes</p>
<p>Dissection blades</p>
<p>Sterile petris</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.