eamag/protocols-with-steps huggingface dataset:
TITLE: P5ARP/P10ARP Media Preparation
AUTHORS: rebecca.bennett

[DESCRIPTION]
How to make P5ARP and P10ARP media plates, simplified from https://wiki.bugwood.org/PARP_or_PARP

Contains: pimarcin, ampicillin, rifampicin, pentachloronitrobenzens. 
Pimaricin is a broad-spectrum antifungal antibiotic that inhibits the true fungi but not most members of the Pythiaceae. Once in solution, it is inactivated by light so that culture plates must be stored and incubated in the dark. Growth of true fungi indicates the inactivation of pimaricin. Concentrations above 10 mg a.i./liter inhibit oospore germination of Pythium and Phytophthora

Plates will last for 1-1.5 weeks before antibiotics are degraded.

[BEFORE_START]
See Safety Warnings.

[STEPS]
SECTION: Preparation
1. Sign up for Autoclave and turn on.

SECTION: Preparation
2. Add 17 g of Difco cornmeal agar to each 2 L Erlenmeyer flask

SECTION: Preparation
2.1. Add RO water to 1000 mL line (pre-measure line and mark with sharpie or use a graduated cylinder).
Cover top with foil so that at least 2" of foil is below flask lip.

SECTION: Preparation
2.2. Add stir bar and mix well over stir plate.
SECTION: Preparation
3. Autoclave media in flask alongside 1 L media bottle (loosely capped), for 20 minutes on liquid cycle.
SECTION: Preparation
4. Turn on hot water bath and set to 50 °C. 

SECTION: Preparation
5. Prepare hood by wiping down with 80% EtOH and labeling Petri dishes.
SECTION: Preparation
6. Prepare rifampicin solution: 0.01 g rifampicin dissolved in 1 mL DMSO in a microcentrifuge tube. 
Cover tube with foil and keep away from light.

SECTION: After Sterilization
7. Check temperature of hot water bath (between 50 °C to 55 °C).
Place sterilized basal media in hot water bath and wait for temperature to decrease target range. Higher temperatures will inactivate antibiotics!

SECTION: After Sterilization
8. Fume hood: 
Add to each 1 L media bottle (wear gloves, eye protection, and dust mask!) on a stir plate: 
0.25 g ampicillin sodium salt
5 mg = 0.005 g Delvocid (50% natamycin)
 

10 mg = 0.01 g rifampicin dissolved in 1 mL DMSO   
1 g Terraclor WP (wettable powder)

SECTION: After Sterilization
9. Mix completely.
SECTION: Plating
10. Pour half into the sterile 1L media bottle.
SECTION: Plating
10.1. Immediately pour into Petri dishes.
SECTION: Plating
10.2. Refill with remaining media and dispense into Petri dishes.
SECTION: Plating
10.3. Immediately fill empty bottle with tap water when finished so that agar does not dry on glass.
SECTION: Plating
11. Mark plates with a single red bar using a Sharpie. Attach lab tape with date and your initials to stack of plates.
SECTION: Plating
11.1. When agar has solidified (8-24h), place back into sleeves and label bottom outside of bag with date made and your initials.
SECTION: Plating
11.2. Store in fridge as soon as possible to preserve the antibiotics.
SECTION: Plating
12. Wash bottles before media dries.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Reagent List:</strong><br />
<ul>
<li>Cell Staining Buffer (BioLegend Cat. No. 420201)</li>
<li>Red Cell Lysis Buffer (BioLegend Cat. No. 420301)</li>
<li>7-AAD Viability Staining Solution (BioLegend Cat. No. 420403)</li>
<li>TruStain FcX™ (anti-CD16/32, BioLegend Cat. No. 101319)</li>
<li>Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301)</li>
</ul>
<br /><strong>References:<br /></strong>Current Protocols in Cytometry (John Wiley &amp; Sons, New York), Unit 6 Phenotypic Analysis.<strong><br /></strong>

[STEPS]
?. 
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?.
TITLE: Neuromelanin staining (Fontana-Masson staining)+ TH-DAB staining on midbrain organoids
AUTHORS: michela.deleidi, Federico   Bertoli, Hariam Raji

[DESCRIPTION]
Fontana-Masson staining is a silver staining technique that is commonly used to identify melanin-containing cells. We combined it with DAB-TH staining that works by using an antibody to detect the presence of TH, followed by a reaction with a substrate (DAB) that results in the formation of a brown-colored product at the site of the antigen-antibody interaction.

[STEPS]
SECTION: Neuromelanin staining
1. Incubate human midbrain organoid sections in a fresh solution of 3:1 methanol (MeOH)/3% hydrogen peroxide at Room temperature for 20 min.

SECTION: Neuromelanin staining
2. Wash the slides and block it.
SECTION: Neuromelanin staining
3. Apply primary antibodies in NGS 5% in PBS+ Triton-X 0.2% solution 60 min at 4 °C. 

SECTION: Neuromelanin staining
4. Next, wash slides. 
SECTION: Neuromelanin staining
5. Prepare ABC solution from Vectastain according to the manufacturer’s instructions (VECTASTAIN® Elite® ABC-HRP Kit, Peroxidase (Standard) PK-6100) and apply to sections for 60 min at Room temperature. 
SECTION: Neuromelanin staining
6. Wash the slides. 
SECTION: Neuromelanin staining
8. Apply DAB solution to the sections at Room temperature for 30 seconds to 12 min depending on when the visible reaction occurred. 

SECTION: Neuromelanin staining
9. For the visualization of neuromelanin, use the Fontana-Masson stain kit, according to the manufacturer’s instructions.  (Fontana-Masson Stain Kit; Sigma‒Aldrich-HT200). 
SECTION: Neuromelanin staining
10. Eventually mount slides with synthetic resin.
SECTION: Neuromelanin staining
2.1. Wash the slides in PBS for 5 min. (1/3)

SECTION: Neuromelanin staining
2.2. Wash the slides in PBS for 5 min. (2/3)
SECTION: Neuromelanin staining
2.3. Wash the slides in PBS for 5 min. (3/3)
SECTION: Neuromelanin staining
2.4. Then, block with NGS 10% in PBS+ Triton-X 0.2% for 60 min at Room temperature.

SECTION: Neuromelanin staining
4.1. Wash the slides in PBS for 5 min. (1/3)
SECTION: Neuromelanin staining
4.2. Wash the slides in PBS for 5 min. (2/3)
SECTION: Neuromelanin staining
4.3. Wash the slides in PBS for 5 min. (3/3)
SECTION: Neuromelanin staining
4.4. Apply secondary antibodies to NGS 5% in PBS+ Triton-X 0.2% solution for 60 min at Room temperature.
SECTION: Neuromelanin staining
7. Prepare DAB solution according to the manufacturer’s instructions by diluting in 50 mL of 1x PBS with 50 µL of 3% H2O2. 

SECTION: Neuromelanin staining
6.1. Wash the slides in PBS for 5 min. (1/3)
SECTION: Neuromelanin staining
6.2. Wash the slides in PBS for 5 min. (2/3)
SECTION: Neuromelanin staining
6.3. Wash the slides in PBS for 5 min. (3/3)
TITLE: Protein extraction and BCA assay from MCAS sorted cells
AUTHORS: Daniel Manrique-Castano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol allows proteins extraction from cells sorted employing the MACS Adult Dissociation Brain kit (Miltenyi Biotec).</div></div>

[STEPS]
?. [Protein Extraction]
Following final centrifugation after MACS cells sorting, discard the supernatant and place the cells  to prevent protein degradation.
on ice
?. [Protein Extraction]
Prepare NP-40 lysis buffer and add it to the cells according to the number of expected proteins.
Usually   -   when protein concentration is expected to be low, or   -   when protein concentration is expected to be high.
?. [Protein Extraction]
Resuspend the cells by constant pipetting, and if it has not been done, transfer the cells to  Eppendorf tubes.
1.5 mL
?. [Protein Extraction]
It is recommended to perform sonication to facilitate protein lysis. Conditions may vary according to the sonication device. Usually, 20-40 seconds, 2 cycles, and middle power (50-70) are enough.
?. [Protein Extraction]
Subsequently, freeze the proteins and keep them at  or
-20 °C
-80 °C
?. [Protein Extraction]
If required, cells can be centrifugated at  and the supernatant must be taken. The pellet is discarded.
Centrifuge: 13000 33, 15 min, 20 10
?. [BCA essay]
The following procedure describes protein measurement by BCA assay using . The protocol must be adapted if a different kit is employed.
?. [BCA essay]
Prepare or thaw BSA standards according to company protocol. Use NP-40 based lysis buffer as blank
?. [BCA essay]
Working reagent must be mixed (50:1, Reagent A:B). To calculate the total required amount, consider the number of replicates for BSA standards and cell samples.   of working reagent re needed per sample/wheel.
200 µl
?. [BCA essay]
In a 96-wheel plate, add   (when the available sample is limited, range limited to 125-2000 ug/ML) or  of standards and samples (achieved range 20-2000 ug/ml).
10 µl
25 µl
?. [BCA essay]
Immediately after, add  of Working reagent to each wheel.
200 µl
?. [BCA essay]
Place the plate in a shaker for  , and subsequently, incubate the plate at  for  .
37 °C
If the microplate reader has a shaker option, placement in a shaker can be avoided and performed in this device.
?. [BCA essay]
Perform protein measurement according to reagent and microplate reader instructions.
Special attention should be paid to the wavelength for absorbance measurement. Some reagents work better at 562, while others at 750. Check kit datasheet.
TITLE: SPOT1 revised protocol
AUTHORS: Guanhua Xun, Huimin Zhao, stlane2 

[STEPS]
?. Using the first provided microcap, collect a saliva sample into capillary A, containing QuickExtract DNA Extraction Solution (Lucigen).  Insert the capillary into the SPOT1 device and press the "Start" button to run the 5-minute pretreatment.
95 °C
?. After pretreatment, remove capillary A from the SPOT1 device and use the second provided microcap to transfer a small volume of pretreated sample to capillary B, which contains the SPOT assay mastermix.  Dispense the pretreated sample into only the top layer of the capillary, as shown in the diagram below.  Disturbing the wax dividing layer during sample transfer may lead to a failed reaction.SPOT assay master mix:  ABCD1       Initial concentration    Final concentration    Amount (μL)  2  <b style="mso-bidi-font-weight:
  normal">Upper compartment  3  WarmStart® Bst 2.0    8000 units/mL  160 units/mL  2  4  WarmStart® RTx    15,000 units/mL  150 units/mL  1  5  Isothermal amplification buffer    10X  0.5X86  dNTPs    10 mM  0.7 mM  5.6  7  MgSO4    100 mm  4 mM  3.2  8  N gene primer mix    10X  0.25X  2  9  E gene primer mix    10X  0.25X  2  10  Saliva samples              5  11  Non-CRISPR nuclease    5 mg/mL or 55 μM    1.375 uM    2  12  MnCl2    50 mM    0.5 mM    0.8  13  Non-primer oligos (total 6 oligos)    100 μM    625 nM    3  14  Reporter probe 1    100 μM    156.25 nM    0.125  15  Reporter probe 2    100 μM    312.5 uM    0.25  16  Nuclease-free water            44.02517  Total            80
ABCD1       Initial concentration    Final concentration    Amount (μL)  2  <b style="mso-bidi-font-weight:
  normal">Upper compartment  3  WarmStart® Bst 2.0    8000 units/mL  160 units/mL  2  4  WarmStart® RTx    15,000 units/mL  150 units/mL  1  5  Isothermal amplification buffer    10X  0.5X86  dNTPs    10 mM  0.7 mM  5.6  7  MgSO4    100 mm  4 mM  3.2  8  N gene primer mix    10X  0.25X  2  9  E gene primer mix    10X  0.25X  2  10  Saliva samples              5  11  Non-CRISPR nuclease    5 mg/mL or 55 μM    1.375 uM    2  12  MnCl2    50 mM    0.5 mM    0.8  13  Non-primer oligos (total 6 oligos)    100 μM    625 nM    3  14  Reporter probe 1    100 μM    156.25 nM    0.125  15  Reporter probe 2    100 μM    312.5 uM    0.25  16  Nuclease-free water            44.02517  Total            80
2 µl
1 µl
8 µl
5.6 µl
3.2 µl
0.8 µl
?. Result ("Positive"/"Negative"/"Inconclusive") will be displayed on SPOT1 device LCD screen after completion of detection reaction and the 1-minute cooling period.
?. Insert capillary B into the SPOT1 device and press the "Start" button to initiate the 35-minute detection reaction.
63 °C
98 °C
TITLE: 5. Metagenomics of Parkinson’s disease implicates the gut microbiome in multiple disease mechanisms
AUTHORS: Haydeh Payami

[DESCRIPTION]
The protocol details the final product , the published manuscript with step-by-step methods. It describes the whole process from start to end using the article "Metagenomics of Parkinson’s disease implicates the gut microbiome in multiple disease mechanisms".

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Humus is a complex mixture of heterogeneous organic compounds derived from plant and animal residues and is formed by the reaction of decomposition, synthesis,&nbsp;and polymerization. It is an integral part of soil organic matter and can be extracted by neutral salts or alkali separating humic and nonhumic fraction. .

[GUIDELINES]
<strong>Materials</strong> <br /><br />Extracting solution: Dissolve 44.6 g of sodium pyrophosphate and 4 g NaOH in 1 L water to maintain 0.1 M strength of the solution.

[STEPS]
?. 
?. 
?. 
?.
TITLE: SOAP-denovo-trans assembly
AUTHORS: Eric J. Carpenter, Naim Matasci, Shuangxiu Wu, Jing Sun, Jun Yu, Fabio Rocha Jimenez Vieira, Chris Bowler, Richard G. Dorrell, Matt Gitzendanner, Ling Li, Wensi Du, Kristian Ullrich, Michael S. Barker, James H. Leebens-Mack, Gane Ka-Shu Wong

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SOAP-denovo-trans assembly protocol used to assemble plant transcriptomes for the 1KP project. </div></div>

[STEPS]
?. SOAPdenovo2 based DBG construction and Trinity based low frequency k-mer removal.
?. Map reads to the contigs and build linkages.a) Utilizing single-end read informationb) Utilizing paired-end read informationc) Merge the linkages
?. Remove erroneous and redundant linkagesa) Manage ambiguous contigsb) Linearize contigs into scaffolds
?. Divide graph into sub-graph. Oases based graph traversal.
?. Gap closure and output the transcripts.
TITLE: Crop FVC retrieval
AUTHORS: Liang Wan, Jiangpeng Zhu, Xiaoyue Du, Jiafei Zhang, Xiongzhe Han, Weijun Zhou, Xiaopeng Li, Jianli Liu, Fei Liang, Yong He, Haiyan Cen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the method of crop fractional vegetation cover (FVC) retrieval from the PROSAIL-GP model. This method can be applied to UAV-based multispectral images to accurately estimate crop FVC in oilseed rape, rice, cotton and wheat.</div></div>

[STEPS]
?. Make sure you have MATLAB 2014b (or newer)  installed on your computer.
?. Dowload the MATLAB scripts from https://github.com/WanLiangZJU/Crop-FVC-retrieval
?. Please use the code in the file of PROSAIL_5B_GP. Here, we provide five main codes for different datasets as follows: Main_oilseed_rape_2017_2018.m, Main_oilseed_rape_2019.m, Main_rice_2018.m, Main_wheat_2019.m, Main_cotton_2019.m
?. Please update the code and run the main codeupdate the file for the input of reflectance dataset the range of model parametersobtain the retrieval result, namely FVC, realted to different leaf angle distributions As for different datasets, it is critical to set the suitable range of mode parameters based on previous studies and field measuremnts.
?. This code uses the UAV-based multispectral image with the spectral region of 604-872 nm. If you use different spectral regions, please refer to the original code onlinehttp://teledetection.ipgp.jussieu.fr/prosail/.It is necessary to modify the range of spectral reflectance in the model to fit with your data.
TITLE: Pregnancy pillow interventions to support the pelvis, lower back, gravid uterus and a left lateral recumbent maternal sleeping position: A rapid systematic review protocol
AUTHORS: Delyth Wyndham, Gwenllian Wynne-Jones

[DESCRIPTION]
Objective: 
The objective of this rapid review is to provide a systematic overview of the literature on the use of pregnancy pillow interventions during pregnancy to support the pelvis, lower back, gravid uterus and left lateral recumbent maternal sleeping position.

Introduction: 
Symptoms and complications experienced during pregnancy are often individualised.  They can vary over time and in intensity during the antenatal period.  The prevalence rates of physiological pregnancy symptoms or complications highlights the need for interventions that ideally address multiple issues.  It is unclear whether pregnancy pillow interventions offer support for pregnancy-related pelvic girdle pain; pregnancy-related lower back pain; abdominal ligament/muscle weakness; and/or facilitate a left lateral recumbent maternal sleeping position. 

Inclusion criteria: 
This review will consider cross-sectional, longitudinal, prospective or retrospective studies; reviews of any type; and syntheses featured in peer reviewed journals or developed by highly regarded organisations.  Outcomes will include supporting the pelvis; supporting the lower back; supporting the gravid uterus; and/or supporting a left lateral recumbent maternal sleeping position.

Methods: 
The review aims to find published research studies, reviews of any type and syntheses (practice or evidence-based recommendations or practitioner guidelines).  The key information sources to be searched are the Cochrane Central Register of Controlled Trials (CENTRAL); EBSCO; and EMBASE (Ovid).  Two reviewers will screen the titles and abstracts for inclusion.  The full text of publications meeting the inclusion criteria will then be examined for eligibility.  Meta-data will be extracted using a standard template.  

Eligible publications will be critically appraised using the relevant JBI Critical Appraisal Tool as appropriate to the publication type.  Reviewer two will evaluated the assessment made by reviewer one in line with rapid review practice.  The results will be synthesised as a narrative synthesis of the publications.
 
Keywords: Rapid Systematic Review Protocol; Pregnancy Pillow Interventions; Pelvis; Lower Back; Gravid Uterus; Maternal Going-To-Sleep Position; Left Lateral Recumbent Sleep.


[STEPS]
TITLE: SOP and tick-sheet for using TDP-43 RNA aptamer (TDP-43APT) to detect pathological TDP-43 in FFPE-preserved human tissue, as described in Spence and Waldron et al., 2024 (Acta Neuropathologica)
AUTHORS: Holly Spence, Fergal M Waldron, Jenna Gregory

[DESCRIPTION]
Here we provide a SOP to outline the correct procedures for performing Immunohistochemistry (IHC) to detect pathological TDP-43 in FFPE-preserved human tissue using the TDP-43 RNA Aptamer (TDP-43APT), as described in Spence and Waldron et al., 2024 published in Acta Neuropathologica (in press at the time of release of this SOP on
01/03/2024)

Users with access to Sequenza immunostaining racks and histological facilities (with fume hood) should be able to carry out all steps over two days.

This protocol uses the TDP-43APT sequence published in Zacco et al., 2022. The sequence is: CGGUGUUGCU with a  3' Biotin-TEG modification, purified using HPLC, scale: 1.0 µM synthesis.
 
Reference for citations of this method
RNA aptamer reveals nuclear TDP-43 pathology is an early aggregation event that coincides with STMN-2 cryptic splicing and precedes clinical manifestation in ALS.
Holly Spence*, Fergal M. Waldron*, Rebecca S. Saleeb, Anna-Leigh Brown, Olivia M. Rifai, Martina Gilodi, Fiona Read, Kristine, Roberts, Gillian Milne, Debbie Wilkinson, Judi O’Shaughnessy, Annalisa Pastore, Pietro Fratta, Neil Shneider, Gian Gaetano Tartaglia, Elsa Zacco, Mathew H. Horrocks, Jenna M. Gregory (2024). Acta Neuropathologica (in press at the time of release of this SOP on 01/03/2024).
*equal contributions, ‡corresponding author

[BEFORE_START]
Please see appendix for materials required to carry out this protocol.

[GUIDELINES]
This protocol is designed for users with access to Sequenza immunostaining racks and histological facilities (with fume hood) should be able to carry out all steps over two days.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Detailed protocol for chemical +/- heat antigen retrieval on sectioned tissue to increase reactivity and specificity of antibody recognition</p>

[GUIDELINES]
<p>Digital pressure cookers give the most consistent results for antigen retrieval on histological slides thanks to programmable, regulated heating without boiling.</p>
<p> </p>
<p>We just have the standard size non-fancy one (it's the size of an instant pot, but fewer cooking options). This InstaPot should work well.</p>
<p><a href="https://www.amazon.com/Instant-Pot-Multi-Use-Programmable-Packaging/dp/B00FLYWNYQ/" target="_blank">https://www.amazon.com/Instant-Pot-Multi-Use-Programmable-Packaging/dp/B00FLYWNYQ/</a></p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/suzcz36.png" data-src="https://s3.amazonaws.com/pr-journal/suzcz36.png" data-ofn="Screen Shot 2018-01-31 at 10.54.22 AM.png" /></p>
<p> </p>

[STEPS]
?. 
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?. 
?. 
?. 
?. 
?. 
?.
TITLE: protocol to use the  anaerobic workstation 
AUTHORS: dollwin.matharu 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">this is a self written protocol to use the anaerobic hood</div></div>

[STEPS]
?. [entering the chamber]
Optional- Open the side chamber from its front door and put in the tray with the materials one wants to take inside, close the front door. Also, make sure that the other materials one needs are already inside
?. [entering the chamber]
Switch on the light, and the foot control device. Also, select the option of side chamber gassing if one performs step 1. The side chamber becomes anaerobic in 4 to 5 minutes, and the bar on the screen indicates it (both boxes become green). If one wants to take tiny objects inside, one can also use the glove door sleeves or small pass tray.
?. [entering the chamber]
Now, enter the hood by inserting one’s hand in the gloves sleeves, make sure one is already wearing their own gloves by this time. The glove door locks are still closed. Perform the vacuum/gas cycle, using the foot control 3 times, and only then open the glove door locks.
?. [working in the chamber]
Now that one is inside, wipe the surface with the wipes. Open the side chamber by pressing the blue button only once (the button is on the right wall). Take the tray inside and unload one’s materials, if the tray is too heavy to pull, use the white curved pulling rod to pull it in. Close the door by pushing the blue button continuously.
?. [exiting the chamber]
To get one’s hands out of the hood, gently pull the hands back (but do not pull them out of the hood at this stage!), lock the glove door using the screw knobs, check its lock by pushing the door gently and pull out one’s hands.
?. [exiting the chamber]
Once completely out, switch off the foot control. Open the black plastic knobs of the glove door to again to check if the screw knobs are locked properly.
?. [exiting the chamber]
Take the tray out of the side chamber by opening its front door and close the door by tightening the knobs. Dispose of the waste. Check all the black knobs on the hood before leaving, their vertical orientation indicates they are closed properly.
?. [exiting the chamber]
Make the entry on the clipboard. Mention one’s initials, working time, pressure of both gases, etc.
?. [working in the chamber]
After one is done, wipe the working area with oxivir wipes. One can use the side chamber tray to take out any waste or any material that should not lie inside the chamber, but remember to close the side chamber door.
?. [working in the chamber]
At this stage, one can begin their work.