eamag/protocols-with-steps huggingface dataset:
TITLE: Experiment Data Depot (EDD) Data Import 
AUTHORS: Jennifer Gin, Christopher J Petzold

[DESCRIPTION]
This protocol details how to import data into an EDD Study. Data can be shared with collaborators via exported CSV files or through the REST API.

For more information about the EDD visit: Experiment Data Depot Google Site.  

Citation: 
Morrell, William C., et al. "The experiment data depot: a web-based software tool for biological experimental data storage, sharing, and visualization." ACS synthetic biology 6.12 (2017): 2248-2259. (link to publication)

Example Links:
https://public-edd.agilebiofoundry.org/
https://public-edd.agilebiofoundry.org/s/example-data-quality-study-2/overview/



[BEFORE_START]
The following instructions show you how to import data for proteomics and metabolomics. 

Create an EDD study (EDD Study Creation protocol) before you start the data import process.

[STEPS]
1. Click the Import Data button on the top right.


 




2. Go through the set of data import steps:

Identify what protocol was used.   Click Shotgun (Discovery) Proteomics.
Select the layout of your data.  Click Generic.

Click Start Import on the bottom right.


 


 

3. Example data layouts:

Here, we have measured the amount of protein "XYLA_ECODH", metabolite "D-Limonene", and extracellular "D-Glucose" for sample "181-aceF" in triplicates at time "12" hours.
 
 
  

 
  

 
  


Drag-and-drop to upload data file into the dropzone box.  


 

4. After the EDD interprets the file it will calculate the number of records to save. Check that this is correct, then click the Save button.  

 

 


5. Once the data is uploaded, the EDD will bring you to the Data page in the Line graph view.  You can toggle to the Bar Graphs.

 


 



 


6. Repeat the Data Import process for other data types.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Emotions are signaled by complex arrays of face and body actions. The main point of contention in contemporary treatments is whether these arrays are discrete, holistic constellations reflecting emotion categories, or whether they are compositional -- comprised of smaller components, each of which contributes some aspect of emotion to the complex whole.  We address this question by investigating spontaneous face and body displays of athletes, and place it in the wider context of human communicative signals and, in particular, of language.  In an experiment, participants interpreted a range of emotions and their strengths in pictures of athletes who had just won or lost a competition.  By matching participants’ judgements with minutely coded features of face and body, we find evidence for compositionality.  The primary bifurcation in this competitive physical context is between Dominance and Submissiveness. The distribution of participants’ responses indicates that other features of face and body cluster to reflect more particular emotional components related, for example, to Positive valence or Goal Obstruction. Therefore, the interaction of the broader and more particular components gives rise to more complex emotional states, such as Happy or Angry in the case of Dominance, and Ashamed or Sad in the case of Submissiveness. </p>

[STEPS]
?. 
?.
TITLE: Flow cytometry-based measurement of trafficking of Golgi to the lysosome via autophagy V3
AUTHORS: Harper JW, Kelsey Hickey, sharan_swarup

[DESCRIPTION]
Protocol for flow cytometry-based measurement of Golgi trafficking to lysosomes via autophagy using Keima-YIPF3/4 reporters in HEK293 cells.

[STEPS]
SECTION: Generation of stable Keima cell lines
1. pLenti vectors expressing either Keima-YIPF4, Keima-YIPF3, or GALNT2-Keima were packaged in 293T cells and the lentivirus was used to infect WT or FIP200-/- HEK293 cells (for Keima-YIPF3 and Keima-YIPF4). GALNT2-Keima was used to infect WT, FIP200-/- and DKO (YIPF3-/-, YIPF4-/-) HEK293 cells. To create stable cell lines, infected cells are selected in 1 µg/mL of puromycin for 48 hours, followed by 48 hours of recovery.
SECTION: Seeding of Keima reporter cells
2. Wash HEK293 cells expressing Keima-YIPF3, Keima-YIPF4, or GALNT2-Keima with 1x PBS
SECTION: Seeding of Keima reporter cells
3. Add Trypsin to cells for 5 min and incubate at 37°C to dissociate cells from plastic well
SECTION: Seeding of Keima reporter cells
4. Resuspend cells in 1 mL DMEM media
SECTION: Seeding of Keima reporter cells
5. Count cells
SECTION: Seeding of Keima reporter cells
6. Seed appropriate number of cells into 96-well, 48-well, or 24-well plates.
SECTION: Seeding of Keima reporter cells
7. Culture cells and analyze for Keima flux with cells <70% confluent. In some cases, induction of flux via nutrient stress is analyzed. The cells were washed twice with PBS and resuspended in DMEM or EBSS to initiate nutrient stress for the indicated time period 12-16 hours, typically). 


SECTION: Keima flux analysis
8. After treatment/starvation, cells were treated with trypsin and quenched with Phenol red free-DMEM. Cells were filtered through a cell strainer cap tube. 
SECTION: Keima flux analysis
9. Use dual-excitation (405nm for ph7 and 561nm for pH3) and collect in 620 nm range using an Attune NxT (Thermo Fisher). Analyze at least 10,000 single, healthy cells. Calculate of acidic:neutral mt-Keima ratio on a per-cell basis in FlowJo Software and plotted using GraphPad Prism.
Optional: include BafA-treated sample (3 hours prior to analysis), to use as normalization for sample set in genotype. Otherwise, fed control samples can be used.
TITLE: Support Protocol 1: Installing MIDAS2
AUTHORS: miriam.goldman  , chunyu.zhao  

[DESCRIPTION]
MIDAS2 is written in Python 3 and can be executed on a 64-bit Linux system. MIDAS2 and its dependencies need to be pre-installed in order to run the commands described in the basic protocols. We recommend the conda package manager for installing these. Alternatively, users who want to ensure reproducibility by using a container with MIDAS2 and its computational environment may use Docker.

[STEPS]
1. Installing Midas2 has 2 options Conda and Docker
2. Install Miniconda
 

3. Configure conda channel.
 

4. Install MIDAS2
 

2. If Docker is properly installed on the system, users can also use the pre-built Docker container.
 

5. Verify your installation.
TITLE: Electroporatic transformation of Rhodobacter sphaeroides
AUTHORS: Jaya  K Yakha, Deborah K Hanson, Rosemarie Wilton, laible

[DESCRIPTION]
The expression host for R. sphaeroides DrshI wasconstructed by deleting Type II restriction enzyme RshI (locus tag RSP_3759; recognition sequence CGATCG) from strain DD11 is suitable for transformation by either chemical or electrical procedures.

[STEPS]
1. 
In a microfuge tube on ice, mix 40 µL  cells with 100 ng of plasmid DNA.

2. 
Turn on the electroporator and select the voltage to 2500 V with other setting of 25uF and 400 ohm.

3. Transfer the cells-DNA mixture to a chilled  electroporation cuvette on ice.

5. Return the cuvette to ice and add 1ml GYCC immediately; record the time constant and the voltage achieved for each pulse.

6. Transfer cells/GYCC from electroporation cuvette to a Falcon 2059 culture tube (15ml). Incubate at 35 °C with slow shaking ( ) for a 240 minoutgrowth period. 

7. Pellet the cells in microcentrifuge tube and spread onto GYCC agar containing appropriate antibiotic. Incubate plates at 33 °C for 2-4 days.

4. Transfer the cuvette to the pulse chamber and shock to desired number of pulses (only once) (if doing multiple pulses, do first pulse and return cuvette to ice for one minute before doing next (and additional) pulses. 1 mine on ice between pulses.
TITLE: Immunological detection of APP and proteins of the endolysosomal system 
AUTHORS: Hankum Park, Frances V Hundley, Harper     JW

[DESCRIPTION]
Here we present a general protocol for immunological detection by Western blotting of APP and proteins of the endolysosomal system, including EEA1, RAB5, PSEN1, LAMP1, LAMP2, TMEM192, and BACE1.  

[STEPS]
SECTION: Western blotting
1. Lyse cell pellets by homogenization in KPBS buffer, urea buffer, or RIPA buffer with protease and phosphatase inhibitors. For some experiments, we employ 293 cells or 293EL APPSw,T700N cells expressing 3xFLAG-EEA1, TMEM192-3xHA, and APP harboring Swedish and T700N mutations as described in dx.doi.org/10.17504/protocols.io.byi7puhn.
SECTION: Western blotting
2. Determined total protein concentration by BCA or Bradford assay.  Normalize samples within a set of samples with additional lysis buffer.  Add NuPAGE LDS buffer (4X) plus NuPAGE reducing agent (10X).
SECTION: Western blotting
3. Load samples onto 4-12% NuPAGE Bis-Tris gels (ThermoFisher), and separate by electrophoresis in MES buffer.
SECTION: Western blotting
4. Transfer proteins to PVDF or nitrocellulose membranes by standard wet transfer in 20% methanol.
SECTION: Western blotting
5. Stain membranes with REVERT 700 total protein stain following manufacturer’s instructions, and image total protein with a ChemiDoc MP (Bio-Rad) at 680 nm.
SECTION: Western blotting
6. De-stain with REVERT reversal solution for 5 min. Block membranes with tris-buffered saline (TBS) plus 5% non-fat dry milk at room temperature for 30-60 min.
SECTION: Western blotting
7. Incubate membranes overnight at 4ºC with primary antibody solution in TBS with 0.1% Tween-20 (TBST) and 5% non-fat dry milk. Wash six times with TBST for 5 min each.  Incubate in secondary antibody solution in TBST (plus 0.01% SDS and 5% non-fat dry milk) for 1h at room temperature.
SECTION: Western blotting
8. Wash membranes four times with TBST for 5 min each.
SECTION: Western blotting
9. When using HRP-conjugated secondary antibodies (Bio-Rad or Cell Signaling Technology), apply luminol and hydrogen peroxide solution to membrane for 2 min and image membrane with a ChemiDoc MP using the chemiluminescent setting.
SECTION: Western blotting
10. When using Li-Cor fluorescent secondary antibodies, blot membranes dry and image with a ChemiDoc MP at either 800 nm or 680 nm, depending on the secondary antibody.
TITLE: siRNA Electroporation of Hydra
AUTHORS: Adrienne Cho

[STEPS]
?. Wash animals (fed one day earlier) 5 times with Milli-Q water and incubate for 45 minutes at 18°C
?. Place 20 animals into electroporation cuvetter with 0.4cm gap and remove as much liquid as possible.
?. Add 200 µL sterilized 10mM HEPES (pH7.0) containing 4µM siRNA
?. Tap cuvette 10 times to evenly distribute animals, leave animals for 5 minutes to allow them to relax and extend.
?. Set Biorad GenePulser Xcell electroporation system to the following: Voltage: 150 VLength: 50 msNumber of Pulses: 2Pulse Intervals: 0.1 s
?. Electroporate animals. Immediately after administering each pulse, add 500 µL 18°C restoration medium.
?. Carefully transfer animals to a dish filled with restoration medium
?. Let animals recover in restoration medium for 48 hours.
?. Repeat Steps 1-8 2x.
TITLE: HotSHOT genomic DNA extraction
AUTHORS: FishFloorUCL, FishFloorUCL

[DESCRIPTION]
How to extract genomic DNA from larvae or finclips using the HotSHOT method.

[STEPS]
SECTION: Materials
1. Prepare the BASE stock solution (50X):
14.03 g KOH crystals (1.25M final concentration)
4 mL of 0.5M EDTA (10 mM final concentration)
ddH2O to 200 mL total volume
Can be stored at room temperature for up to four years
SECTION: Materials
2. Prepare the NEUTRALISATION stock solution (50X)
63.04 g Tris-HCL (2M final concentration), also called Trizma HCl
ddH2O to 200 mL total volume
Can be stored at room temperature for up to four years
SECTION: Procedure
3. Prepare fresh 1X BASE and 1X NEUTRALISATION solution in nuclease-free H2O.
SECTION: Procedure
5. Add 50 µL of 1X BASE solution into each well of the plate

SECTION: Procedure
6. Seal the plate and place on PCR block 95 °C 30 min 
SECTION: Procedure
7. Cool at Room temperature 

SECTION: Procedure
8. Add 50 µL of 1x NEUTRALISATION solution into each well of the plate.

SECTION: Procedure
9. Note, the extracted DNA concentration is often too high for downstream applications like PCR or KASP.
SECTION: Storage
10. Store at 4 °C if you will use the DNA in the next few weeks. Beware, the samples will slowly evaporate from a sealed plate. Alternatively, -20 °C for long-term storage. This will also prevent the samples from evaporating.
SECTION: Procedure
4. Transfer finclips or culled larvae to individual wells of a 96-well plate.
TITLE: Calcium fluorimetry with the FLIPR Calcium 6 kit on FlexStation 3
AUTHORS: Angus Li, Samuel Liu, Rennica Huang, Seungkirl Ahn, Robert J Lefkowitz

[DESCRIPTION]
This protocol details an experimental procedure used to generate results described in the manuscript Li, A., Liu, S., Huang, R., Ahn, S., & Lefkowitz, R. J. (2023). Loss of biased signaling at a G protein-coupled receptor in overexpressed systems.

[STEPS]
1. Plate U2OS-TetOn-AT1R in microplates (Black with clear bottom, lysine- coated Corning 3842) at 15000 cells/well 2 days prior to assay
2. Add doxycycline and optionally PTX to wells 14 hours before replacement with loading buffer
3. Remove one vial of Calcium 6 Assay Reagent (Component A, good for 2 plates) from the freezer and equilibrate to room temperature.
4. Dissolve contents of one Component A vial by adding 10 ml of 1X HBSS Buffer plus 20 mM HEPES. Mix by vortexing (~1-2 min) until contents of vial are dissolved. It is important that contents are completely dissolved to ensure reproducibility between experiments.
5. Remove cell plates from the incubator, Calcium 6 Kit - add an equal volume of loading buffer to each well in the dark (i.e. 120ul HBSS+HEPES + 40ul of Dye/well (4X) for a 96-well plate in the original protocol). Use 11.8ml of HBSS+HEPES +4.8ml of Dye (4X) for one plate. Save the dissolved dye solution in -20.
6. Return plates to the incubator and incubate two hours at 37 C, 5% CO2
7. Optionally, add YM-254890 1 hour prior to reading
8. 30 minutes before experiment (1.5 hours into incubation), prepare the reagents (5X, 40ul/well+20ul volume). The ligand needs to be plated in a 96 well with round bottom (The FlexStation will add ligand, shake, and measure sequentially).
9. Turn on FlexStation 30 minutes before experiment and warm up to 37.
10. Place tips and ligand plate
11. After the two-hour incubation period, transfer the assay plate directly to the FlexStation instrument assay plate carriage 10 minutes prior to reading to re-equilibrate temperature, and then run the assay.
12. In an individual well or column of wells, the calcium flux peak(s) should be complete within 1 to 3 minutes after addition. For an entire plate however, the protocol will not complete until all chosen columns are finished. 40-45 min for entire 96 well plate.
TITLE:   ELISA for quantification of IL-7 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>Bone marrow stromal cells produce IL-7 that acts on pre-B cells and T cells. It causes B-cell and T-cell proliferation.</span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-7 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl  of human serum. Human IL-7 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-7 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-7 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-7 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-7 standard dilutions and the human IL-7 sample concentration determined.
?. For better results place the microplate on a plate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The presence and structure of EFNs in <em>Opuntia robusta</em> had not been investigated. We used light, scanning-electron, and transmission-electron microscopy to examine morphology, anatomy, and ultrastructure of the secretory spines in areoles in female and hermaphrodite individuals of <em>O. robusta</em>. Young cladodes develop areoles with modified and secretory spines as EFNs only active during the early growth phase of female and hermaphrodite individuals. EFNs are non-vascularized structures, with no stomata, that consist of three distinct tissues: a basal meristematic tissue; a middle elongation region; and an apical secretory cone (asc) formed by large globular epidermal cells, of sac shape, containing nectar and medullar elongated cells.</p>

[STEPS]
?. 
?. 
?.
TITLE: KAPP-Sen TMC: Dissociation of Pancreatic Islets (non-recovered)
AUTHORS: Juliana Alcoforado Diniz, Jessica Garofalo, Dylan Baker, Paul Robson

[DESCRIPTION]
The dispersed samples were shipped cold from PRODOLABS. Prior to scRNA-seq dispersed samples from brain dead donor’s pancreatic islets were dissociated as follows.

[STEPS]
SECTION: Cell Dissociation with Accutase
1. 
 

SECTION: Fixation of Cells &amp; Nuclei for Chromium Fixed RNA Profiling
2. Cells were fixated prior to scRNAseq according to https://dx.doi.org/10.17504/protocols.io.x54v9py5zg3e/v1
SECTION: Cell Dissociation with Accutase
1.1. Transfer cell suspension of pure islets to a new 50ml tube. Use additional media to rinse original container.
SECTION: Cell Dissociation with Accutase
1.2. Centrifuge at room temperature 130g for 5 mins.
SECTION: Cell Dissociation with Accutase
1.3. Aspirate the supernatant and add media to the appropriate concentration of 1,000 islets/1 ml.               Ex: If receiving 7,000 islets, use 7 ml media.
SECTION: Cell Dissociation with Accutase
1.4. Add half of the islets (recovery) to the coated flask and put to incubate at 37°C.
SECTION: Cell Dissociation with Accutase
1.5. To the other half of the islets (no recovery), add media to 10 ml and centrifuge then aspirate carefully.
SECTION: Cell Dissociation with Accutase
1.6. Add accutase at a concentration of 1 ml/1,000 islets. Mix with pipette and incubate at 37°C. Coat pipette tip and use to mix every 2 mins, checking at 6 mins.
SECTION: Cell Dissociation with Accutase
1.7. Add CMRL 1066 (Cat. 11530037) to approximately 9 ml media/1 ml accutase then centrifuge at 230 g.
SECTION: Cell Dissociation with Accutase
1.8. Aspirate supernatant and resuspend in 1.5 ml of CMRL
SECTION: Cell Dissociation with Accutase
1.9. Filter through a 40 µm Flowmi.
SECTION: Cell Dissociation with Accutase
1.10. Count cells using AO/PI (acridine orage/propidium iodide) Cell Viability Kit for Luna-FL automated cell counter.
SECTION: Cell Dissociation with Accutase
1.11. Proceed to cell fixation.
TITLE: Anti-iNKT MicroBeads Isolation protocol
AUTHORS: Ajit N Shah, Rachel Hatano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">isolation of 6b11 positive cells </div></div>

[STEPS]
?. Determine cell number.
?. Centrifuge cell suspension at 300×g for 10 minutes. Aspirate supernatant completely.
?. Resuspend cell pellet in 400 μL of buffer per 10⁸ total cells. Add 100 μL of Anti-iNKT MicroBeads per 10⁸ total cells.
?. Mix well and incubate for 15 minutes in the refrigerator (2−8 °C).
?. Wash cells by adding 1−2 mL of buffer per 10⁸ cells and centrifuge at 300×g for 10 minutes. Aspirate supernatant completely.
?. Resuspend up to 10⁸ cells in 500 μL of buffer.
?. Place column in the magnetic field of a suitable MACS Separator.
?. Prepare column by rinsing with the appropriate amount of buffer: LS: 3 mL
?. Apply cell suspension onto the column. Collect flow-through containing unlabeled cells.
?. Wash column with the appropriate amount of buffer. Collect unlabeled cells that pass through and combine with the effluent. LS: 3×3 mL.
?. Remove column from the separator and place it on a suitable collection tube.
?. Pipette the appropriate amount of buffer onto the column.
?. Immediately flush out the magnetically labeled cells by firmly pushing the plunger into the column. LS: 5 mL
?. . To increase the purity of iNKT cells, the eluted fraction is enriched over a second MS or LS Column.