eamag/protocols-with-steps huggingface dataset:
TITLE: HEPES transformation buffer
AUTHORS: Andreas Sagen

[DESCRIPTION]
HEPES buffer is commonly used to prepare prokaryotic (bacterial) cells for electroporation.

[STEPS]
SECTION: 1 M HEPES buffer
1. Add 40 mL distilled water to a 50 mL tube
SECTION: 1 M HEPES buffer
2. Measure and add 11.915 g HEPES

Materials:
 
SECTION: 1 M HEPES buffer
3. Adjust pH to pH 7.4 with concentrated sodium hydroxide or hydrogen chloride
SECTION: 1 M HEPES buffer
4. Add distilled water to 50 mL
SECTION: 1 M HEPES buffer
5. Filter sterilize with a 0.2 µm pore-size filter
TITLE: X2Go Client Set-Up
AUTHORS: Laurel Dieckhaus

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">X2Go Client set-up to run Linux from Windows computer (on and off campus). </div><div class = "text-block">Use the following link for more information or help on setting up X2Go Client:</div><div class = "text-block"><a href="https://helpdesk.engr.arizona.edu/support/solutions/articles/33000204314-how-can-i-access-engineering-linux-systems-from-home-" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://helpdesk.engr.arizona.edu/support/solutions/articles/33000204314-how-can-i-access-engineering-linux-systems-from-home-</span></a></div></div>

[STEPS]
?. [Install X2Go Client]
Install X2Go Client from the following site in the section labeled Get X2Go: https://wiki.x2go.org/doku.php. Choose the correct client install based on your type of computer you have (for example, for Windows, you will choose the Windows Installer).
?. [Define X2Go Client Session]
Open X2Go Client.
?. Select Session in the upper left corner and New Session from the drop-down menu.
?. The program will open a new window where you will enter session preferences.
?. The main parameters used will be: Host, Login and Session Type. Other parameters can be customized to the user but will not be reviewed in this protocol.
?. Type engr-hutchin02g.catnet.arizona.edu in the Host text box.
?. Type your University of Arizona username in the Login text box.
?. Select MATE for the Session Type.
?. Select the Connection tab.
?. It is recommended that Connection Speed be set at WAN when you are on campus using the Linux box. If you are working from home, it is recommended to set this to ADSL.
?. Select the Input/Output tab.
?. It is recommended that you set Display to Custom and set the width and height to 1600x900. The Fullscreen setting will set your entire screen black while the client is loading and can make other items hard to access.
?. Select the Media tab. Have your settings match what is shown below.
?. Shared Folders tab can be modified to include a folder to your current desktop for any data  collection or protocols you run in the X2Go client. This is useful to avoid the need to email or download files to a drive.
?. Once all session preferences have been finalized, click on the session in the right column of the X2Go window.
?. A new login dialog box should appear. Type in your password and press enter. Loading up X2Go client will take a few minutes after logging in.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol provides a method to study phage-bacteria interactions by generating a Network in Cytoscape using the application CoNet. To <a href="http://psbweb05.psb.ugent.be/conet/tutorial5.php" target="_blank">CoNet tutorial</a> can help with analysis. Based on the methods from the following publication:<br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Perl scripts and other supplementary information is available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<strong>Required Software</strong><br />
<ul>
<li>Cytoscape v3.1.1</li>
<li>CoNet</li>
</ul>
<br /><strong>Relevent Files</strong><br />Output:<br />
<ul>
<li>CoNet/skinmet_metaphlan_merged_output_genera.txt</li>
<li>CoNet/taxonomy_species_rel_abund_table.tsv</li>
<li>CoNet/org_skinmet_for_conet.txt</li>
<li>CoNet/org_phage_for_conet.txt</li>
<li>CoNet/org_perm.txt</li>
<li>CoNet/org_boot.txt</li>
</ul>
R script: <a href="https://www.protocols.io/view/Script-R15-CoNet-Analysis-Formatting-Relative-Abun-ejpbcmn" target="_blank">R15</a>

[STEPS]
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TITLE: Transformation
AUTHORS: Zhujun Wei

[STEPS]
?. Put DH5α chemically competent bacterium on ice for melting about 5 min.
?. Add 50 μl DH5α chemically competent bacterium into 1.5 ml tubes, mix with ligation product or digested product thoroughly, incubate the bacterium on ice for 30 min.
?. Heat shock the bacterium for exactly 45 s at 42 ℃ water bath.
?. Place back on ice for 2 min.
?. Add 500 μ l LB medium to each tube, shake the tubes at 37 ℃, 200 rpm for 45 min.
?. Centrifuge the bacterium at 4200 r for 3 min.
?. Discard 300 μl supernatant and resuspend the sediment.
?. Plate 100 μl for a reaction product.
?. Incubate plates upside down overnight at 37℃.
TITLE: BrainSaw agar stock solution
AUTHORS: Rob Campbell

[DESCRIPTION]
Making agar stock solution for serial sectioning imaging.

[STEPS]
SECTION: Before you start
1. You will need at least 100 mL  of 50 mM PB. 
 


SECTION: Make the stock
2. Rinse out the 100 ml bottle in which the stock is kept. 
 

SECTION: Make the stock
3. Fill the bottle with 100 mL of 50 mM PB to the notched fill line.

SECTION: Make the stock
4. Weigh out 5 g of   and pour into the bottle. Cap the bottle and shake it. 

SECTION: Using the stock
5. Do not heat the stock solution bottle. This concentrates it over time and it takes ages to heat. Instead, pour out the desired amount into a beaker and heat carefully in the microwave.  Use low or medium power and stop as it begins to boil or even just before. The agar will look slightly less clear if you don't boil it, but this does not matter.
TITLE: Dispensing agar into multiwell plates
AUTHORS: Bonnie Evans

[DESCRIPTION]
Updated protocol for pouring agar into multiwell plates using Integra VIAFILL dispenser. Agar should be prepared in advance, and kept in 60oC waterbath until ready to dispense, and whilst dispensing. The X, Y, Z positions for dispensing can be adjusted according to the multiwell plates being used. 

[STEPS]
SECTION: Configure Integra VIAFILL
3. Insert large cassette into the machine 
SECTION: Configure Integra VIAFILL
4. Configure X, Y, and Z settings for the multiwell plate by clicking on tool symbol -> stage alignment.
 

SECTION: Configure Integra VIAFILL
5. Exit settings by pressing the back button
SECTION: Dispensing Agar
6. Press on the program you wish to use (see later for configuring your own program)
SECTION: Dispensing Agar
7. Make sure that the correct cassette is listed and change if necessary
SECTION: Dispensing Agar
8. Select the volume you wish to dispense
 

SECTION: Configure Integra VIAFILL
1. Prepare a 250ml bottle of hot milliQ water in the microwave and keep in the waterbath along with the agar. The water is important to have on hand in case of tubing blockages.
SECTION: Configure Integra VIAFILL
4.1. Put the plate into the stage and then press 'Move' so that the plate moves so that it is under the dispensing cassette. 
SECTION: Configure Integra VIAFILL
4.2. Use the up and down arrows to move the pipette tip so that they hover just over the plate and make note of the height (this will be entered into the dispensing program at a later step). Press 'Fast/Slow' button to switch between fast and slow movements.
SECTION: Configure Integra VIAFILL
4.3. Use the X, Y arrows to move the plate so that the pipette tips are centered in the middle of column 5.

SECTION: Configure Integra VIAFILL
4.4. Save all settings.
SECTION: Dispensing Agar
10. Place the end of the tubing from the cassette into the agar that is being kept warm in the water bath
SECTION: Dispensing Agar
11. Press 'Prime' to prime the tubing and allow to finish so that agar flows from the pipette tips.
 

SECTION: Dispensing Agar
9. Select 'set height' and set the appropriate height for tip height (usually all the same)
SECTION: Dispensing Agar
12. Place a clean plate in the stage
SECTION: Dispensing Agar
13. Press run and then plate should fill with agar
SECTION: Dispensing Agar
14. Repeat steps 11-12 until all the plates have been filled.
 

SECTION: Cleaning the cassette
15. 'Reverse prime' all the agar
SECTION: Cleaning the cassette
16. Place the tubing ends into the hot water.
SECTION: Cleaning the cassette
17. Prime so that the water runs through and clears all the agar
SECTION: Cleaning the cassette
18. Reverse prime to remove the water
SECTION: Cleaning the cassette
19. Release tension from the tubing and remove cassette
SECTION: Cleaning the cassette
20. Double wrap the cassette in aluminium foil for autoclaving
SECTION: Configure Integra VIAFILL
2. To pour 6, 12, 24 or 48 well plates, disconnect the appropriate tubing from the cassette. Tube 1 is closest to the instrument. Select 96 well plate in programme.
TITLE: DNA Short-insert Library Construction Protocol for Illumina HiSeq 2500/4000/X Ten or NovaSeq
AUTHORS: Hongfang Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">HiSeq DNA pair-end library construction is used for DNA sequencing on Illumina HiSeq 2500/HiSeq 4000/HiSeq X Ten/ and NovaSeq sequencers. The insert size of the DNA fragments can be 300bp, 350bp and 500bp.</div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/protocols-files/files/dqhebjitx.jpg" /></div></div>

[STEPS]
?. Fragment Genomic DNA  genomic DNA was randomly fragmented using a Covaris ultrasonicator. The fragmented DNAs were tested by Gel-electrophoresis, then purified using an paramagnetic bead based AxyPrep Mag PCR clean up Kit.
1 µl
?. End RepairThe fragmented DNAs were combined with the End Repair Mix, incubated at for .  The end-repaired DNA is then purified with a AxyPrep Mag PCR clean up Kit.
20 °C
?. Add A-Tails to the 3’ end The repaired DNAs are combined with the A-Tailing Mix, incubated at  for . The DNA needs to be purified again with an AxyPrep Mag PCR clean up Kit.
37 °C
?. Adapter Ligation and Pooling Illumina adapters are ligated to the Adenylated 3'Ends of the DNA, incubated at  for . Purify the Adapter-ligated DNAs with AxyPrep Mag PCR clean up Kit. The concentration is measured by a Caliper high-throughput Bioanalyzer.
16 °C
?. Fragments Selection The PCR products are selected by agarose gel electrophoresis with target fragments. Purify the gel with a QIAquick Gel Extraction kit (QIAGEN). Re-dissolve the appropriate amount of Elution Buffer and attach the library tag to complete the library construction.
?. PCR Several rounds of PCR amplification with PCR Primer Cocktail and PCR Master Mix are performed to enrich the Adapter-ligated DNA fragments.
?. Products Purification PCR products are selected by running another 2% agarose gel to recover the target fragments. Purify the gel with a QIAquick Gel Extraction kit (QIAGEN). Re-dissolve the appropriate amount of Elution Buffer and attach the library tag to complete the library construction.
?. Library QC and SequencingLibrary was qualified by the Agilent Technologies 2100 bioanalyzer and ABI StepOnePlus RealTime PCR System. Libraries that pass these QC steps can then be sequenced using pair-ends on the Illumina HiSeq/NovaSeq System.
TITLE: Recovery and preparation for transplantation of cryopreserved vmDA progenitors for transplantation.
AUTHORS: Tyra Fraser, Lachlan Thompson

[DESCRIPTION]
This protocol outlines the recovery of cryopreserved vmDA progenitors. After recovery, this outlines the process to prepare these cells for xenotransplantation. 

[GUIDELINES]
All work is to be conducted in well sterilised laminar flow hoods designated for human iPSC work where possible to minimise contamination.

[STEPS]
SECTION: Recovery of cells
1. Prepare NBB27 base media according to the materials table.

3.  Aspirate supernatant. Flick pellet twice.
 Resuspend in 1 mLof NBB27 + All + Ri 1:1000.
Pipette 10 µL into a small Eppendorf tube for cell counting. Repeat for a second Eppendorf tube.
Take the 2 tubes and add 10 µL trypan blue to cells in each tube. 
Place 10 µL of mixed cells in haemocytometer.
Count cells in each quadrant.
Calculate total number of cells. Repeat this for tube 2 to ensure an accurate cell count.

Total cells = Average count of quadrants x Dilution factor x Volume (ml) x 10^4

8.  Calculate the total volume needed to resuspend cells to a final density (typically between 100-150K/µl.  
9. Spin cells(300 x g, 3 min, 4 °C)





SECTION: Recovery of cells
2. Place5 mL of NBB27 media + All + Ri 1:1000 in a 15 mL falcon tube. 
Warm cryopreserved cells in hand or in a water bath until a small chunk of ice is remains
 Remove thawed portion into the tube containing the 5 mL (prepared in as per Step 1)
Take 500 µL of media and use it to thaw the remaining chunk of ice.
Remove all media from cryopreserved tube and place into the falcon tube with media.  
Spin cells(300 x g, 3 min, 4 °C). 
 




4. Label small Eppendorf tube with specific details about the transplant.
Aspirate supernatant
Add half the required media on top of the cells gently (i.e. if you have 2 million cells total final volume is 20 µL to achieve 100 000 cells/uL, so add 10 µL of media
(NBB27 + All + Ri 1:1000) to pellet.   
Using a P20, gently disturb the pellet in a circular motion, taking care not to damage cells
by hitting the edge of the tube with the pipette tip. 
Once mixed, take up the suspension once or twice and transfer to a small Eppendorf tube. 
Measure the volume of cell suspension. 
Add the appropriate volume of NBB27 + All + Ri 1:1000 required to reach the final volume. 
Place cells on ice ready for use
TITLE: hmbc_metab.nan
AUTHORS: NAN KB, John Glushka, Mario Uchimiya, Christopher Esselman, Saraa Al Jawad, Leandro I Ponce, Laura Morris, Arthur Edison

[DESCRIPTION]
This is a protocol for running the Bruker pulse program "hmbcetgpl3nd" for metabolomics samples.

[BEFORE_START]
This protocol assumes:
Your sample is loaded, locked, tuned for both proton and carbon channels, and shimmed in the magnet
The calibrated 90° pulse value for proton (i.e., P1) for the sample has been collected

[GUIDELINES]
This protocol intends to provide concise instructions to carry out the experiment. For more comprehensive information, see Bruker's documentation "Basic NMR Experiments" by clicking ? →  Manuals (docs) on the menu bar on TopSpin. See also "Pulse Program Catalogue. 1D/2D" for the details about the pulse program used in this protocol.

[STEPS]
SECTION: Create a new dataset
1. 
SECTION: Create a new dataset
1.1. On the menu bar on TopSpin, click on
Start → Create Dataset
 

 

SECTION: Create noesypr1d experiment file
0.23. When Lock, Tune, Shim are complete proceed to the specific protocol for your desired pulseprogram(s).
SECTION: Create a new dataset
1.2. Enter
NAME: Name of a set of datasets (e.g., human_serum_study1). Use a single string.
EXPNO: Dataset number. Use a positive integer.

Select
Directory: Your directory.
 

 

3. 
SECTION: Create a new dataset
1.3. Select
Read parameterset

Click the button
Select
SECTION: Create a new dataset
1.4. A new window opens. On the right top bar, select
Source = /opt/NAN_METAB/par
 
In the list, select the one you want to use:

For serum and plasma samples:
HMBC_NUS_br600_serum.par: Parameter set using an acquisition mode "non-uniform sampling (NUS)". Higher resolution on the indirect dimension

For urine samples:
HMBC_NUS_br600_urine.par: Parameter set using an acquisition mode "non-uniform sampling (NUS)". Higher resolution on the indirect dimension

 

Click
OK
SECTION: Acquire a spectrum
2. Go to the "USE DEFAULT" tab below to proceed with the default optimized parameters.
3.1. Load the calibrated P1 using the following command in the command line.
 
(e.g., getprosol 1H 10.01 -7.45)

 

3.2. Click on
Acquire → Gain
in the menu bar to automatically set the receiver gain.
 

 

3.3. Click
Go
in the menu bar to acquire a spectrum.
 

3.4. After the run, click on
Process → Proc. Spectrum
in the menu bar to execute an automated processing macro.
 

3.5. If you want to modify parameters to improve your spectrum,  and move to the step case "MODIFY PAR".
SECTION: Acquire a spectrum
3. 
SECTION: Acquire a spectrum
3.1. Select the AcquPars tab to show a parameter set.
 

 

SECTION: Acquire a spectrum
3.2. Click the icon
_∏_
to see a complete parameter set.

 

SECTION: Acquire a spectrum
3.3. Change parameters as needed.

Typically, the main parameter to adjust is NS:
 

SECTION: Acquire a spectrum
3.4. Other potential parameters to adjust: 
 

 

SECTION: Acquire a spectrum
3.5. Other potential parameter for the NUS parameter sets:
 

SECTION: Acquire a spectrum
3.6. Acquire a spectrum. If more optimization is needed,  .

 

SECTION: Create a new dataset
1.5. Click
OK
TITLE: ImageJ Analyses of Mitochondria (HSP60) in TH+ cells
AUTHORS: Chiara Pavan, Clare Parish

[DESCRIPTION]
This protocol explains how to use Fiji to analyse mitochondria in iPSC derived dopaminergic neurons in culture for parameters such as network branching, sphericity and mean total branch length.

[BEFORE_START]
Requirements prior to analysis
-  Install Fiji
-  Locate images to analyse (HSP60+TH staining)
-  Install Mitochondria Analyzer Plugin (see references)

[STEPS]
SECTION: Image acquisition
1. Images analysed with this protocol were taken with Leica Stellaris 8 confocal (for mitochondrial morphology and ATG13-HSP60 co-localization, with FLIM 40x/1.30 oil immersion objective, HC PL
APO, CS2, using Leica Power HyD detector S and HyD detector X).

All mitochondria morphology images were acquired in 3D by optical sectioning under Lightning™ mode with a z-stack of 1.73μm and maximum voxel size of 94.7 nm laterally (x, y) and 350 nm axially (z). For each experimental sample 10 cells per sample were analyzed. 

Note: Deconvolved images were used for the mitochondrial morphology analysis.
SECTION: Analysis pipeline
2. 1. Open the image

 

2. In the TH channel (use the bar under the figure to go to the TH channel, here channel 2), use the Freehand Selection tool to mark the cell of interest
 
If the TH signal is not bright enough, go to Image>Adjust > color balance (only in the TH channel, don’t modify the other channels)

3. Save the region of interest selected by going to Analyze > Tool > ROI manager > Add [t]

4. Duplicate the HSP60 channel by clicking outside the selected ROI, then going to Image > Duplicate; select Channel 1 and tick hyperstack

5. Clear the staining outside the ROI by selecting the ROI (press on the ROI name on the ROI
manager panel), then going to Edit >Clear Outside >Yes

6. Convert the image to 8 bit by going to Image>Type>8bit

7. To optimize the Threshold, go to Plugins > Mitochondria Analyzer > 3D Threshold Optimize (for more info, see below) 
NOTE: This may take some time

 

8. Once decided the optimal values, go back to your picture (the one with only HSP60, duplicated, cleared), and apply the chosen parameters by going to Plugins > Mitochondria Analyzer > 3D Threshold and inserting the desired parameters.
NOTE: This may take some time
TITLE: Mouse Whole Cell Tissue Processing for 10x Genomics Platform
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes how to isolate tissue from various ROI’s of the mouse brain followed by preparation of a highly concentrated single cell suspension for transcriptomic profiling on 10x Genomics platform.

Note: 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.

[STEPS]
TITLE: Conexão do actígrafo no computador - ActTrust - V.1
AUTHORS: Daniel Vartanian

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Este protocolo faz parte do processo de coleta de dados actigráficos do Grupo Interdisciplinar de Pesquisa em Sono (GIPSO). Ele foi desenhado para os actígrafos provenientes da companhia </span><a href="https://www.condorinst.com.br/" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Condor Instruments</span></a><span>. Algumas das configurações mencionadas podem ser específicas desses aparelhos.</span></div><div class = "text-block">Se você não é membro ou parceiro do GIPSO, você pode utilizar este protocolo como referência para o processamento de seus equipamentos. Neste caso, por favor, não envie os formulários mencionados neste ou em outros protocolos relacionados ao processo.</div><div class = "text-block"><span>Se você utilizar os protocolos do GIPSO em sua pesquisa, por favor considere citá-los na seção de materiais e métodos de seu estudo. Nós nos esforçamos bastante para elaborar e manter protocolos de acesso aberto à comunidade científica. Você pode encontrar a citação deste protocolo na aba</span><span style = "font-weight:bold;"> metadata</span><span>.</span></div><div class = "text-block"><span>Consulte a aba </span><span style = "font-weight:bold;">Guidelines</span><span> para visualizar o fluxograma do processo e os demais protocolos relacionados.</span></div><div class = "text-block"><span>Para dúvidas ou sugestões, entre em contato pelo e-mail </span><a href="mailto:gipso@usp.br" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">gipso@usp.br</span></a><span> .</span></div></div>

[STEPS]
?. Abra o software ActStudio
Caso apareça algum pop-up solicitando a configuração da latitude e longitude, clique em Não. Para este procedimento essa configuração não é necessária.
?. Acesse a tela Controle
Na parte superior do ActStudio você verá um botão chamado Controle. Clique sobre ele e depois selecione a aba CONTROLE.
?. Conecte o ActDock (base de conexão) no computador
Conecte a ponta USB A-Type (a ponta com o conector maior) do cabo de conexão no computador e a outra ponta do mesmo cabo no ActDock.
?. Conecte o actígrafo no ActDock
Pegue o actígrafo e conecte-o no ActDock, cuidando para que os contatos dourados do aparelho estejam alinhados aos contatos dourados da base de conexão.Feche o ActDock até ouvir um "click" (sem pressionar demais o actígrafo contra a base). Após isso, o registro do actígrafo deve aparecer na aba CONTROLE do software ActStudio.ATENÇÃOApós concluir esta etapa pode ocorrer que actígrafo não conste na aba CONTROLE. Isso normalmente se dá por uma falha de contato. Neste caso, tente adicionar um peso sobre o actígrafo com o ActDock aberto.Quando o actígrafo está com a bateria esgotada é possível que ocorra um erro ao conectar. Se esse for o caso, ignore o erro e aguarde alguns minutos com o aparelho conectado. Assim que o actígrafo receber alguma carga ele será identificado pelo sistema.