eamag/protocols-with-steps huggingface dataset:
TITLE: Transformation of Chemically Competent Cells
AUTHORS: NUS iGEM

[STEPS]
?. Add  of miniprep plasmid or  of Gibson product into competent cell
1 µl
5 µl
?. Incubate cells in  water bath for
42 °C
?. Incubate cells on ice for
?. Add  of LB media into cell sample
1 ml
?. Incubate cells at  for
37 °C
?. Spin down the cells at  for
Centrifuge: 6000 34
?. Remove  of the supernatant
700 µl
?. Re-suspend pellet in remaining media
?. Transfer  of the culture onto the agar plate containing appropriate antibiotic
100 µl
?. Spread the cells evenly
?. Incubate at  overnight
37 °C
TITLE: FR-Match: cell type matching for scRNAseq data
AUTHORS: Yun Renee Zhang, Brian Aevermann, Richard Scheuermann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">FR-Match is a supervised cell phenotype matching strategy for cluster-to-cluster cell transcriptome integration across scRNAseq experiments.  </div><div class = "text-block"><span>An R package and Shiny application are provided at </span><a href="https://github.com/JCVenterInstitute/FRmatch" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/JCVenterInstitute/FRmatch</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Data preparation and exploration]
Use the built-in data preparation function to create data objects with required and optional input data elements. Create data objects for experiment 1 (E1) and experiment 2 (E2)
?. [Data preparation and exploration]
View comparative cell cluster sizes.
?. [Data preparation and exploration]
View "barcode" plot for cluster of interest.
?. [Run main algorithm]
Use wrapper function to perform bi-directional matching.
?. [Run main algorithm]
Map E1 to E2.
?. [Run main algorithm]
Map E2 to E1.
?. [Combine and plot matching results]
Combine the bi-directional matching results and plot.
?. [Additional plots]
Some optional plotting functions to help studying the matching results.
?. [Additional plots]
Plot one-directional matching results.Plot one-directional matching p-values.
?. [Additional plots]
Minimum spanning tree (MST) plot. MST can be plotted by turning on the plot option in the test function.
?. [Launch Shiny app]
Interactively explore and match scRNAseq cell type clusters with the seamless Shiny app. The Shiny app may serve as a quick start demo with pre-loaded datasets.
TITLE: Pole Test
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Test for motor ability in mice

[STEPS]
1. Place the wooden, 50 cm long, 1cm in diameter the home cage to be tested, and make sure it stands upright on stand.
2. Set up recording camera to record the trials.
3. Habituate the mice to the pole one time before testing by carefully placing the mouse on the top of the pole facing downwards. Allow the mouse to travel down and reach the home cage.
4. After habituation, test the mouse by again placing them on the top of the pole facing downwards.  Allow the mouse to travel down and reach the home cage. There is no time cut-off.
5. Record the time that the mice took to descend the first half, the second half, and the total pole.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>ADAMTS5, the main aggrecanase, is a protein that seems to play a functional role in the development of brown and white adipose tissue (WAT) and browning of WAT. These published observations were made using the ADAMTS5-P mice originally generated by Pﬁzer, in collaboration with Lexicon Genetics (The Woodlands, TX, USA).</p>

[GUIDELINES]
<p>-Tails digested in lysis buﬀer containing proteinase K can be stored at -20°C before continuing the protocol.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Micro Volume Purification of His-Tagged Proteins with IMAC Ni-Charged Resin
AUTHORS: Syon Schlecht, Emily Gunderson, Ruthie Fowler, Takara Aguilar

[DESCRIPTION]
This protocol describes a micro volume purification method for His-tagged proteins using IMAC with Ni-charged resin in a mini spin chromatography column. His-tagged proteins are selective bound to a Ni-charged resin due to the interaction between the polyhistidine tag and Ni²⁺ ions and eluted using imidazole-containing buffers. Desalting is the performed using SEC where salts and imidazole are fractionated, while larger molecules like the target protein are excluded. This results in the isolation of purified His-tagged proteins with a high yield and minimal contaminants, suitable for downstream applications.




[STEPS]
SECTION: Preparing and Equilibrating Column
1. Resuspend   and pipette 200 µL  into a    

SECTION: Preparing and Equilibrating Column
3. Wash the   by adding 200 µL of distilled water

SECTION: Preparing and Equilibrating Column
2. Centrifuge the  at 1000 x g, 2 min and discard any collected buffer 

SECTION: Preparing and Equilibrating Column
4. Centrifuge the  at 1000 x g, 2 min and discard any water collected

SECTION: Preparing and Equilibrating Column
5. Equilibriate the column by adding 200 µL of   to the   
 



SECTION: Preparing and Equilibrating Column
6. Centrifuge the  at 1000 x g, 2 min and discard any buffer collected and cap the column

SECTION: Sample Binding and Elution of His-Proteins
7. From the soluble fraction of lysed cells containing the    add  600 µL to the    and gently mix for 20 min 

SECTION: Sample Binding and Elution of His-Proteins
8. Centrifuge the  at 1000 x g, 2 min and discard any flowthrough 
SECTION: Sample Binding and Elution of His-Proteins
9. Add 600 µL of   to the    and centrifuge at 1000 x g, 2 min and discard any collected buffer 
 



SECTION: Desalting of His-Sample
11. Snap the bottom and remove the top of a   and allow the packing buffer to drain for 5 min 
 


SECTION: Desalting of His-Sample
12. Centrifuge the  at 1000 x g, 2 min and discard any collected buffer 

SECTION: Desalting of His-Sample
13. Add the approx. 100 µL of the collected   to the   
 

SECTION: Desalting of His-Sample
14. Centrifuge the   at 1000 x g, 5 min and any excluded liquid is the purified   

SECTION: Desalting of His-Sample
15. Repeat up to 3 times with the same   to obtain the desired   volume

SECTION: Sample Binding and Elution of His-Proteins
10. Add 100 µL   to the   and centrifuge at 1000 x g, 2 min , and COLLECT the flowthrough, containing the
TITLE: Behavioral tests in rodents coupled with dopamine signaling manipulations
AUTHORS: Chuyu Chen, Loukia Parisiadou

[DESCRIPTION]
These two protocols describe behavioral tests associated with Parkinson’s disease phenotypes in mice. The striatal motor learning protocol (rotarod test) is a two-phase paradigm assessing striatal motor learning under reversible dopamine antagonism. 

[STEPS]
SECTION: Rotarod test
1. General Rota-rod test is performed as follows: 
SECTION: Rotarod test
1.1. The rotarod apparatus (Panlab LE8205, Harvard Apparatus) equipped with a mouse rod (3 cm diameter)
SECTION: Rotarod test
1.2. Set the rotarod with a start speed of 4 rpm, and set to 4–40 rpm acceleration over 300s
SECTION: Rotarod test
1.3. Hold the mouse by tail. Place the mouse on the rod and face away from the direction of rotation.
SECTION: Rotarod test
1.4. Start acceleration and record the time for each mouse to fall.
SECTION: Rotarod test
1.5. Perform five trials with intertrial-interval = 15 s, max trial duration = 300 s (one daily session)
SECTION: Rotarod test
1.6. Calculate the average time to fall over five trials.
SECTION: Details of dopamine signaling manipulations for Rota-rod
2. The specific task consists of eighteen daily sessions divided into two phases (Figure 1). 
SECTION: Details of dopamine signaling manipulations for Rota-rod
3. Administer intraperitoneally dopamine receptor antagonists for sessions 1-5 30 min prior to testing prepared in 0.9% saline): cocktail of 1 mg/kg SCH23390 + 1 mg/kg eticlopride, 1 mg/kg SCH23390, or 1 mg/kg eticlopride.

SECTION: Details of dopamine signaling manipulations for Rota-rod
4. Following a 72-hr break, test the mice for another thirteen sessions (drug-free recovery phase)
SECTION: Open field test
5. The open field arena (56 x 56 cm) is used to assess locomotor activity.
SECTION: Open field test
6. Turn on the lights in the recordingg chamber. Clean the arena with 70% ethanol and adjust the camera border to match the arena edge. 
SECTION: Open field test
7. Wait for 5min to allow the ethanol to evaporate completely prior test.
SECTION: Open field test
8. A mouse is placed in the center of the arena and its ambulation activity is collected by the LimeLight software for 20 minutes.
SECTION: Open field test
9. Clean the arena with 70% ethanol between tests. 
SECTION: Open field test
10. The LimeLight software provides the total distance traveled and the percentage of time/distance within different parts of the arena.
TITLE: qRT-PCR sample preparation
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for qRT-PCR sample preparation for analysis using a RotorGeneQ machine (Qiagen). 

[STEPS]
1. Synthesise cDNA libraries from total RNA from each sample using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), using Oligo(dT)20 primers.
2. Place the synthesised libraries on ice, and dilute the libraries 1:3 by adding 60 µL of DEPC-treated H2O to each sample, pipetting up and down gently to mix ~ 8 times.

3. Dilute primer stocks by adding 2 µL of the forward primer, and 2 µL of the reverse primer, to 46 µL of DEPC-treated H2O on ice to make a final combined forward and reverse primer stock of 4 micromolar (µM) forward primer and 4 micromolar (µM) reverse primer for each gene target to be analysed. 

4. Thaw the 2x QuantiNova SYBR green master mix (Qiagen) on ice.
5. Depending on the number of samples being analysed, assemble a sample master mix (not containing the cDNA library) containing the following:


 

6. To qRT-PCR tubes sitting on ice (Qiagen – RotorGeneQ compatible), add 6 µL of the master mix for each target gene to the desired tubes, and add 4 µL of the appropriate cDNA library to each sample.
7. Tap the rack holder to ensure all solution is at the bottom of the tube before capping each sample.(NOTE: samples do not need to be mixed prior to analysis)
8. Load and run the samples on a RotorGeneQ (Qiagen) machine using the desired parameters for the gene targets you are analysing.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes a methond of single nucleus isolaton from human skeletal muscle for snRNA sequencing.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Coffee Protocol
AUTHORS: John  Borghi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is intended as a demonstration of what I like to call the </span><span style = "font-weight:bold;">Coffee Cup Rule</span><span> - Document more than you think is necessary and with greater specificity that you think is necessary.</span></div><br/><div class = "text-block">The name is derived from the fact that the cup measures on drip coffee machines refer to "cups of coffee" a unit of measure that is between 4 and 6 fluid ounces rather than a "cup", which is 8 fluid ounces in the United States. Because of this, it is important to be very specific when describing the amounts needed to make a pot of coffee. </div><br/><div class = "text-block">The directions here will make a good enough cup of coffee, but a truly replicable protocol would require even more specificity about the roast of the coffee, the grind of the coffee, the temperature of the water, the make and model of the drip coffee machine, etc. The amounts and directions would also be different if using other methods for brewing coffee. If you're using a French Press, you'd probably want a slightly different coffee-to-water ratio than what's described here.</div></div>

[STEPS]
?. [Make Coffee]
Measure out  and . If the scale is out of batteries, 4 1/4 cups (US) of water and 8 tablespoons of coffee will work in a pinch.  Use a measuring cup for the water not the cup measures on the side of the coffee pot. Those refer to "cups of coffee".
[water]
[ground coffee]
?. [Make Coffee]
Pour the water in the drip coffee machine. Pour the coffee into the filter and put the filter into the machine. Brew on "Bold" setting, if available.
?. [Make Cup of Coffee]
Pour   and  in a mug.Enjoy!
[milk]
[brewed coffee]
TITLE: Amicon NMWCO Filter Concentration and Buffer Exchange
AUTHORS: Lauren  Adams

[DESCRIPTION]
This Amicon NMWCO filter protocol helps concentration dilute elution fractions containing protein as well as assists with buffer exchange if necessary. 

[STEPS]
1. Wash the filter three times with Optima grade water and centrifuging at 14,000 x g. Equilibrate the column by washing once with the same buffer as the elution buffer containing the target protein for ~10-30 minutes or until the volume has decreased.
2. Load the elution fraction containing the target protein into the filter and centrifuge at 14,000 x g for ~10-30 minutes or until the volume has decreased. Repeat this until the total elution volume has decreased ≤ 25 µL for LC-MS or ≤ 80 µL for I2MS. If the buffer is already acidic (TFA or FA), no additional buffer exchange is required. However, if the elution buffer is not acidic (ex: 100 mM Tris Base), buffer exchange should be performed. 
3. Gradually incorporate larger volumes of 0.2-0.5% TFA or FA to the sample volume after sequential centrifuge cycles until the majority of the final buffer is TFA or FA. Remove sample from filter by centrifuging upside down at 14,000 x g into a clean LoBind tube. Bring the final volume up to 25 µL for LC-MS or 80 µL for I2MS with 0.2-0.5% TFA or FA before analyzing by mass spectrometry.
TITLE: https://www.facebook.com/ViaKetoGummiesEnPharmacieFrance/
AUTHORS: Via Keto En Pharmacie France

[DESCRIPTION]
Via Keto En Pharmacie France

[STEPS]
1. https://www.facebook.com/ViaKetoGummiesEnPharmacieFrance/
https://www.facebook.com/ViaKetoEnPharmacieFrances/
https://www.facebook.com/Via-Keto-Gummies-En-Pharmacie-France-101317322656200
https://via-keto-gummies-en-pharmacie-france.jimdosite.com/
https://lexcliq.com/via-keto-en-pharmacie-france-reaction-choquante-via-keto-en-pharmacie-france-arnaque-et-cout-ou-puis-je-acheter/
https://via-k-e-t-o-en-pharmacie-france.jimdosite.com/
https://sites.google.com/view/via-keto-enpharmacie-france/
https://groups.google.com/g/via-keto-en-pharmacie-france/c/68RLSYimDKg
https://www.cos.youth4work.com/Via-Keto-En-Pharmacie-France/jobs/174965-via-keto-capsules-avis-clients-en-pharmacie-et-acheter-en-france-in-Ile-de-France
https://twitter.com/ViaPharmacie/status/1545834023553048577
https://techplanet.today/post/via-keto-en-pharmacie-france-us-reviews-top-1-nice-via-keto-en-pharmacie-france-capsules-examine-before-purchase
https://www.scoop.it/topic/via-keto-en-pharmacie-france-official-by-via-keto-en-pharmacie-france-official/p/4133866601/2022/07/09/via-keto-en-pharmacie-france-reviews
https://techplanet.today/post/via-keto-en-pharmacie-france-clients-en-pharmacie-et-acheter-en-france
https://www.riverdavesplace.com/forums/threads/https-www-facebook-com-viaketogummiesenpharmaciefrance.255047/
https://kukooo.com/https-www-facebook-com-viaketogummiesenpharmaciefrance/
https://keithburgun.net/forum/index.php?threads/https-www-facebook-com-viaketogummiesenpharmaciefrance.398/
https://keithburgun.net/forum/index.php?threads/https-www-facebook-com-viaketogummiesenpharmaciefrance.399/
https://kenhmuare.net/threads/https-www-facebook-com-viaketogummiesenpharmaciefrance.26217/
https://www.iphoneforums.net/threads/https-www-facebook-com-viaketogummiesenpharmaciefrance.85291/
https://community.dimensionplus.in/d/265-httpswwwfacebookcomviaketogummiesenpharmaciefrance
TITLE: BASIC PROTOCOL 2: Download MIDAS Reference Database
AUTHORS: miriam.goldman  , chunyu.zhao  

[DESCRIPTION]
This protocol describes how to download all or part of a MIDASDB, a set of custom files constructed from microbial genome sequences and containing all the information needed to metagenotype the species detected in a set of shotgun-metagenomic samples. MIDAS2 provides two prebuilt MIDASDBs sourced from large, public microbial genome collections: MIDASDB-UHGG (4,644 species / 286,997 genomes) based on the Unified Human Gastrointestinal Genome catalog (v1) [9] and MIDASDB-GTDB (47,893 species / 258,405 genomes) based on the Genome Taxonomy Database (v202) [10]. Support Protocol 2 describes how to build a new MIDASDB locally from a custom genome collection. A MIDASDB should be downloaded or built before any other MIDAS2 protocols can be run.


There are three components in a MIDASDB: single-copy marker genes (SCGs), representative genomes (rep-genome), and pangenomes (pan-genome). Each species contributes sequences to all three components. By preloading the MIDASDB, individual calls to MIDAS2 commands do not need to automatically download the necessary files. As a result, with a preloaded MIDASDB, per-sample analyses can be run in parallel without a risk of processes interfering with one another.

[STEPS]
1. Initialize a local copy of MIDASDB-UHGG

 

 


2. Customize the MIDASDB download. In Basic Protocol 1, 22 species were present in at least one sample list_of_species.tsv). Now those will be downloaded in the database components (both rep-genome and pan-genome) only for these 22 species.
 

3. The download has completed successfully when the command midas2 database --download finishes and no error is reported.
TITLE: Red Velvet Cupcakes 
AUTHORS: Hannah Gunderman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Photograph by Alexandra Kusper on Unsplash.</div><div class = "text-block"><span>Note: this recipe is adapted from </span><a href="https://choosingchia.com/vegan-red-velvet-cupcakes" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://choosingchia.com/vegan-red-velvet-cupcakes/</span></a><span>. </span></div></div>

[STEPS]
?. Preheat the oven to 350 degrees F and line a cupcake pan with cupcake liners.
?. Mix the almond milk and apple cider vinegar and set aside for 5 minutes to turn to “buttermilk.”
?. Next, add the coconut oil and vanilla extract.
?. Mix all the dry ingredients together in a bowl.
?. Pour the wet ingredients into the dry ingredients and mix together until smooth. If using vegan food colouring, add it now.
?. Spoon the batter into your pan, filling 3/4 of the way full.
?. Bake for 17-19 minutes, or until a toothpick comes out clean.
?. To make the cream cheese frosting,  remove the can of coconut milk from the fridge and scoop out just the cream on top  (the cream and liquid should have separated in the fridge) (*skip this step if using store-bought coconut whipped cream).
?. Add the vanilla extract and cane sugar and whip on high until smooth and the whipped cream forms soft peaks.
?. Add the cream cheese and continue to whip, just until combined.
?. Use a piping bag to pipe the frosting onto the cupcakes.
?. Serve immediately and enjoy!
?. [Ingredients]
Cupcakes: 3/4 cup almond milk1 tsp apple cider vinegar1/3 cup coconut oil, melted and cooled1  tsp vanilla extract1 cup Spelt flour (or regular flour, or gluten-free flour blend)3/4 cup organic sugar2 tbsp cocoa powder1/2 tsp baking powder3/4 tsp baking soda1/4 tsp salt2 tsp beetroot powder or vegan red food colouringCream cheese frosting: 1 can full-fat coconut milk  (or 2 cups store-bought coconut whipped cream)1 tsp vanilla extract4 tbsp organic sugar1/2 cup vegan cream cheese1/3 cup coconut oil, melted and cooled1  tsp vanilla extract1 cup Spelt flour (or regular flour, or gluten-free flour blend)3/4 cup organic sugar2 tbsp cocoa powder1/2 tsp baking powder3/4 tsp baking soda1/4 tsp salt2 tsp beetroot powder or vegan red food colouringCream cheese frosting: 1 can full-fat coconut milk  (or 2 cups store-bought coconut whipped cream)1 tsp vanilla extract4 tbsp organic sugar1/2 cup vegan cream cheeseCream cheese frosting: 1 can full-fat coconut milk  (or 2 cups store-bought coconut whipped cream)1 tsp vanilla extract4 tbsp organic sugar1/2 cup vegan cream cheese
?. [Preparing and Baking]
Follow the steps listed below for making these (vegan) red velvet cupcakes!
TITLE: Cryo-EM Grid preparation
AUTHORS: Annan SI Cook

[DESCRIPTION]
This protocol details Cryo-EM grid preparation.

[STEPS]
SECTION: Grid Preparation
1. Freshly glow-discharge the QUANTIFOIL R 2/1 mesh Cu 300 holey carbon grids using a glow-discharge system (PELCO easiGlow) at 25 mA current for 1 min.
SECTION: Sample Application
2. Mix the PI3KC3-C1~RAB1A sample with 0.05% n-Octyl-β-D-Glucopyranoside (OG) to achieve the desired final concentration.
SECTION: Sample Application
3. Apply a small volume (typically 3-4 µL) of the sample onto the glow-discharged side of the grids.
SECTION: Vitrification
4. Load the grids with the sample into the Vitrobot cryo-plunger.
SECTION: Vitrification
5. Set the Vitrobot to the following conditions:
SECTION: Vitrification
5.1. Maintain 100% humidity inside the chamber.
SECTION: Vitrification
5.2. Set the temperature to 4 °C.

SECTION: Vitrification
5.3. Wait for 3 s.

SECTION: Vitrification
5.4. Blot using a blotting force of -15 (may need calibration depending on your particular instrument) .
SECTION: Data Acquisition Setup
6. Prepare the Cryo-EM microscope (300 kV Titan Krios) for data acquisition.
SECTION: Data Acquisition Setup
7. Used X-FEG and an energy filter set to 20 eV.
SECTION: Data Collection Parameters
8. Use SerialEM software for automated data collection.
SECTION: Data Collection Parameters
9. Set the following parameters for data collection:

Magnification: 81,000x.
Super-resolution pixel size: 0.525 Å.
Defocus range: -0.8 to -2.2 micrometers.
Collect 50-frame image stacks.
SECTION: Data Collection
10. Initiate the data collection process using the specified parameters.
SECTION: Data Collection
11. Monitor the data acquisition progress and ensure that the images are being recorded.
SECTION: Cumulative Dose Control
12. Each movie has a cumulative electron dose of 50 e/Å².