eamag/protocols-with-steps huggingface dataset:
TITLE: Non-Enzymatic Generation of Placenta Single Cells from Third Trimester Human Placenta
AUTHORS: Ameloko Joy, Idowu Aimola, Fomukong A Hanneda, Reuben B. Samson, Zeenat B Kudan, Habiba H. Abubakar, Musa Kana, Benedict Anchang

[DESCRIPTION]
The placenta is a heterogeneous and complex organ with multiple cell types, posing a challenge for the field of maternal-fetal medicine to implement single-cell technologies for a deeper characterization of this essential organ. Several protocols use enzymes to digest the tissue and generate single cell suspension, but this approach has several shortcomings including the loss and reduced viability of cells. In this study, we describe a non-enzymatic approach to generate single cell suspension
from placental tissue with high yield and viability for single cell RNA
sequencing. 

[STEPS]
SECTION: Placenta Single Cell Preparation Protocol
1. Transform placenta sections into gentle MACs C-tube containing 3000 µL of MACs running buffer.
SECTION: Placenta Single Cell Preparation Protocol
2. Set up a gentle MACS dissociator program to h-cord-01 for 553 rotation per round (rpr) at
30 s.

SECTION: Placenta Single Cell Preparation Protocol
3. After the run, centrifuge at 300 x g for 10 min at 40 °C.

SECTION: Placenta Single Cell Preparation Protocol
4. Remove the supernatant leaving 200 µL to resuspend pellet in. Transfer pipettes may be used
to remove supernatant after centrifugation to minimize loss of placenta cells.

SECTION: Placenta Single Cell Preparation Protocol
5. 10 µL of placental cells can be counted on a countess II cell counter or using trypan blue.
TITLE: GFP-ATG3 GUV Assay 
AUTHORS: lmstrong

[DESCRIPTION]
LC3 lipidation on GUVs

[STEPS]
1. GUV Preparation 
SECTION: GUV Preparation
1. 
1.1. Clean the coverglass
1.2. Coat cleaned coverslips with 100 μL 5% (w/w) polyvinyl alcohol (PVA) with a molecular weight of 145,000 (Millipore). Place the coated coverslip in a heating incubator at 60 °C to dry the PVA film for 30 min.
1.3. Spread a lipid mixture at 1 mg/ml with a molar composition of 70% DOPC, 20% DOPE, 5% DO-PI(3)P, 5% DOPS, 0.3% Atto647N DOPE uniformly onto the PVA film.
1.4. Put the lipid-coated coverslip under vacuum overnight to evaporate the solvent.
1.5.  Use 100 μL 330 mOsm sucrose solution for swelling for 1 h at room temperature
1.6. Harvest the GUVs and use them with 12 h.
2. GUV Assay   
2.1. Set up the reaction in an eight-well observation chamber (Lab Tek) at room temperature.
2.2. Coat the chamber with 1 mg/ml β casein for 10 min.
2.3. Wash the coated chamber with reaction buffer (25 mM HEPES at pH 7.4, 150 mM NaCl and 1 mM TCEP).
2.4. Make a 120 µL reaction mixtures with the proteins and 5 mM ATP and 1 mM MgCl2. The final concentration of WIPI2d 200 nM, E3 complex is 50 nM, ATG7 is 100 nM, GFP-ATG3 or Mutant is 100 nM, and mCherry-LC3B is 500 nM.
2.5. Add 3 µL GUVs to initiate the reaction.
2.6. Pick random views for imaging within 5 min.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The task described here is that used in the PLoS ONE paper entitled 'Continuous Robust Sound Event Classification Using Time-Frequency Features and Deep Learning'. It builds upon the long established standard isolated sound evaluation task first described by Jonathan Dennis, and widely used by a number of other authors - for evaluating classifiers of isolated sounds.</p>
<p> </p>
<p>By contrast the current task extends this to potentially overlapping sounds with no a priori knowledge of start and end points.</p>
<p><br />The advantage of having a standard task is obvious: it makes experiments easily repeatable by others, and eases the comparison of results when other authors make use of the same method to evaluate their own research. With at least 15 state-of-the-art sound classification papers published with Dennis' isolated sound event detection method, it is easily the most popular task defined to date.</p>
<p> </p>
<p>In this current task, exactly the same raw material is used, but is extended through protocol and setup into a continuous, overlapping and robust classification task.</p>
<p><br />The task uses freely available sound recordings from the Real World Computing Partnership (RWCP) Sound Scene Database in Real Acoustic Environments. These must be obtained directly by the RWCP, and are free for non-commercial or academic users, while commercial users are charged a small fee.</p>
<p> </p>
<p>Robustness evaluation is performed by mixing raw sounds with background noises from the NOISEX-92 database at several signal-to-noise (SNR) levels. The NOISEX-92 data is widely available online for download.</p>

[BEFORE_START]
<p><strong>Obtain the required data and tools</strong></p>
<p> </p>
<p>The <strong>RWCP Sound Scene Database </strong><strong>NOISEX-92</strong> recordings of <em>Destroyer Control Room</em>, <em>Speech Babble</em>, <em>Factory Floor 1</em> and <em>Jet Cockpit </em><em>1</em>.</p>
<p> </p>

[GUIDELINES]
<p>All processing will be done in <strong>MATLAB</strong>. <strong>Octave</strong> is a free alternative which works well but will require several minor changes in procedure and operating files.</p>
<p>For classification purposes, a <strong>machine learning toolbox</strong> is recommended (e.g. <a href="https://uk.mathworks.com/matlabcentral/fileexchange/38310-deep-learning-toolbox" target="_blank">Deep Learning Toolbox</a> which is simple for beginners to start with, although has been superseded by tools such as MatConvNet or TensorFlow).</p>

[STEPS]
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?.
TITLE: Login to UA HPC
AUTHORS: Ken Youens-Clark, Bonnie Hurwitz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This describes setting up ssh keys and configuration to avoid having to use your NetID+ 2-factor authentication every time you log into the HPC.  Windows users will need to install a free Unix-like environment that provides a terminal so as to avoid using an ssh client like Putty (for example: Cygwin (http://www.cygwin.com)).  Cygwin users must be sure to install the 'ssh' tools which are not installed by default.</div></div>

[STEPS]
?. Sign up for NetID+ https://webauth.arizona.edu/netid-plus/
?. Initial loginOpen a terminal and type 'ssh @hpc.arizona.edu'.  Enter your NetID password and then follow the instructions for 2-factor authentication. You will be presented with a menu that says:===============HPC.ARIZONA.EDU===============Please select a target system to connect to:(1) Ocelote(2) El Gato(Q) Quit(D) Disable menuSelect "D" to disable the menuDisabling menu...
?. Copy public key to speed up loginOpen a second terminal on your local machine and 'cd ~/.ssh'.  If that directory does not exist, execute 'ssh-keygen' to create local keys; accept all defaults by pressing .  Then copy your public key:  $ cat ~/.ssh/id_rsa.pubCopy and paste that text. On MacOS, you can use the 'pbcopy' (pasteboard copy) command:  $ pbcopy In the HPC terminal from your initial login (@gatekeeper), open '~/.ssh/authorized_keys' and add the line of text from the 'id_rsa.pub.'  If the file does not exist, create it.  Ensure that the permissions on the file are 600 ('chmod 600').
?. Test no-password loginBack in your local terminal, test that you can login without 2-factor auth by executing 'ssh @hpc.arizona.edu'.  If you were logged in automatically, you are all set.
?. Create login aliasIn your local terminal, open '~/.ssh/config' and add the following lines:  Host hpc  Hostname hpc.arizona.edu  User Now on your local machine, you can "ssh hpc" to get a terminal on the HPC.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Guide for reagent/buffer preparation for protocol to separate chloroplasts from diatom cells using ammonium fluoride to permeate the silica frustrule and a percoll gradient to separate the plastid from other cellular components.

[GUIDELINES]
Buffers and reagents can be mixed up ahead of time and stored cold (refrigerated). Percoll gradients (layering of 40% and 80% percoll solutions) should be done the same day of the extraction shortly before use.

[STEPS]
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TITLE: Western Blot CTB
AUTHORS: Arindam Naha

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>For studying intracellular accumulation of CTB subunits in </span><span style = "font-style:italic;">E coli</span><span>, bacteria were grown up to 4 hours following induction by 0.2% of arabinose. Post induction, bacterial cultures were mixed with equal volume of translation-translocation halt cocktail (200 µg/ml chloramphenicol, 200 mM sodium azide, 9.5% ethanol) and immediately placed in an ice water bath for 15 minutes of incubation to arrest protein synthesis and translocation. Cells were harvested by centrifugation at 14000 rpm for 15 minutes at 4°C. Supernatants were preserved and used for CT-ELISA experiment as described before. Pellets were then suspended in TME buffer (20 mM Tris-Cl pH 8.0, 2mM β-mercaptoethanol, 1 mM EDTA), preferably 1/10</span><span style = "vertical-align:super;">th</span><span> volume of the original culture and disrupted by sonication. After centrifugation at 13,000 rpm at 4°C for 15 min to remove any unbroken cells, crude cell lysates were used for measurement of protein concentration using Bradford assay (BioRad, USA). Equal amount (≥80 μg) of protein samples were mounted on to 15% SDS-PAGE. Blots were probed with classical CTB-specific monoclonal antibody (anti-Cla CTB, 1:5000) or anti-beta lactamase (anti-βla 1:500) antibody. A portion of the same blot was stained with Ponceau S staining solution (Sigma-Aldrich, St Louis, MO, USA) to validate whether equal amount protein samples were transferred in the blot. Blots were developed using West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL) according to the manufacturer’s instructions and visualized in ChemiDoc XRS</span><span style = "vertical-align:super;">+ </span><span>system (BioRad, USA). Densitometric analysis of band intensities was performed using the Multi Gauge software V2.3(FujiFilm).</span></div><style>
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[STEPS]
TITLE: Plasmid Construction Protocol
AUTHORS: Xuefeng Ren, Annan SI Cook

[DESCRIPTION]
This protocol details plasmid construction in general terms for the insertion of genes into the pCAG mammalian expression vector.

[STEPS]
SECTION: Design Plasmid Constructs
1. This protocol uses the pCAG vector and NEB restriction enzymes and Gibson Assembly kits.
SECTION: Design Plasmid Constructs
2. Obtain the DNA sequences for the genes of interest from your preferred DNA synthesis vendor.
SECTION: Digestion or Gibson Assembly
3. Decide whether to use restriction digestion or Gibson assembly for subcloning.
SECTION: Digestion or Gibson Assembly
4. Restriction Digestion:
SECTION: Digestion or Gibson Assembly
4.1. Digest both the pCAG vector and the DNA fragments (genes/tags) using appropriate restriction enzymes.
SECTION: Digestion or Gibson Assembly
4.2. Purify the digested fragments using a DNA purification kit.
SECTION: Digestion or Gibson Assembly
5. Gibson Assembly:
Follow the manufacturer's protocol for the Gibson assembly kit to assemble the DNA fragments into the pCAG vector.
SECTION: Ligation
6. If using restriction digestion, perform a ligation reaction to insert the digested DNA fragments into the linearized pCAG vector.
SECTION: Ligation
7. Use a DNA ligation kit and follow the manufacturer's instructions.
SECTION: Transformation
8. Transform the ligated plasmids into competent E. coli cells.
SECTION: Transformation
9. Plate the transformed cells on selective agar plates containing the appropriate antibiotic for plasmid selection.
SECTION: Bacterial Culture
10. Incubate the plates   at an appropriate temperature for E. coli growth (e.g., 37 °C).

SECTION: Plasmid Isolation
11. Pick colonies that have grown on the plates and inoculate them into liquid culture with the same antibiotic.
SECTION: Plasmid Isolation
12. Grow the cultures to obtain a sufficient amount of plasmid DNA.
SECTION: Plasmid Isolation
13. Isolate the plasmid DNA using a plasmid purification kit. 
SECTION: Plasmid Isolation
14. Sanger sequence the results.
TITLE: Highway carbon unlocking efficiency study based on super-efficiency SBM-Malmquist
AUTHORS: Yun Chen, qi luo, machongsen

[DESCRIPTION]
Carbon Neutrality goals and Sustainable Development Goals (SDGs), as new requirements for global development at this stage, have raised higher requirements for achieving the coordination of economic efficiency and ecological development of transportation infrastructure, especially highways. To promote the achievement of Carbon neutrality goals and SDGs, this research intends to investigate the carbon unlocking efficiency of highways. In this paper, we take China as an example, use the data of 18 listed highway companies and their provinces from 2010-2021 to conduct the study, measure the static carbon unlocking efficiency by using the super-efficiency SBM model with undesirable outputs, combine with the Malmquist index model for the decomposition of the efficiency and the dynamic analysis, and use the Tobit regression model to analyze the factors affecting the carbon unlocking efficiency of the highway. The results show that (1)From a static perspective, the highway carbon unlocking efficiency shows a steady upward trend, and the highway carbon unlocking efficiency has been in a state of low efficiency in general, with obvious regional differences. (2)From a dynamic perspective, highway carbon unlocking efficiency changes are relatively flat, highway carbon unlocking efficiency change index is in the rising stage, relying on technical efficiency can effectively improve the level of carbon unlocking efficiency. (3)The regional economic level, industrial structure, and urbanization level have a significant positive correlation with carbon unlocking efficiency, while the level of scientific and technological development and the level of opening up have a negative effect.

[STEPS]
1.
TITLE:        ELISA for quantification of macrophage-colony stimulating factor (M-CSF) in human serum or plasma.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. An anti-human macrophage-colony stimulating factor (M-CSF) coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. M-CSF present in the serum sample binds to antibodies adsorbed into 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-M-CSF 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-M-CSF antibody.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP conjugate.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of M-CSF present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human M-CSF standard dilutions and the human M-CSF sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: U Mass - Chronic/acute phloridzin treatment
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Phloridzin is a potent inhibitor of renal glucose reabsorption and may be used to lower serum glucose levels. Phloridzin may be administered chronically to induce glycosuria and lower hyperglycemia in diabetic mice.  Phloridzin may be administered acutely to reduce serum glucose levels for experiments in diabetic mice. </div></div>

[STEPS]
?. For chronic phloridzin treatment: 1. Anesthetize mice with an intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). 2. Shave hair at the incision site on the back. 3. Make an incision (~0.5 cm) using sterilized scalpel between the scapulae. 4. Subcutaneously insert an Alzet mouse osmotic pump containing phloridzin (0.4 mg/kg body weight). 5. Suture or close the incision site using sterilized staples. 6. Administer ketoprofen to minimize pain and house mice individually. 7. Alternatively, phloridzin may be chronically administered using a twice daily intraperitoneal injection at 0.4 mg/kg body weight.
?. For acute phloridzin treatment:1. Survival surgery is performed to establish a chronic indwelling catheter at 5~6 days prior to experiment for intravenous infusion.  (refer to M1023: Surgery-jugular vein cannulation) 2. Mice are fasted overnight (~15 hours) or for 5 hours prior to the start of experiment. 3. Place a mouse in a rat-size restrainer with its tail tape-tethered at one end. 4. Expose and flush the intravenous catheter using saline solution. Then, connect the catheter to the CMA Microdialysis infusion pump. 5. Phloridzin is intravenously infused at 100 g/kg/min for 60~120 min to lower serum glucose levels in hyperglycemic, diabetic mice.
TITLE: Western Blot (Tank-Blot)
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Blotting Buffer:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">100mL 10x SDS running buffer </li><li style = "counter-reset:ol0;list-style-type:disc;">200mL 100% Methanol</li><li style = "counter-reset:ol0;list-style-type:disc;">700mL H20</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">Fill a tray with blotting buffer</li><li style = "counter-reset:ol0;list-style-type:disc;">Transfer SDS gel to the blotting buffer</li><li style = "counter-reset:ol0;list-style-type:disc;">Assemble blotting sandwich (take it, soak sponge and place on it, soak filter paper and place on it, place inverted SDS gel on top, without creating bubbles.Add charged PDVF membrane with forceps ( charged in 100% ethanol and then soaked in buffer ) </li><li style = "counter-reset:ol0;list-style-type:disc;">Add soaked filter </li><li style = "counter-reset:ol0;list-style-type:disc;">Streak out bubbles</li><li style = "counter-reset:ol0;list-style-type:disc;">Add soaked sponge</li><li style = "counter-reset:ol0;list-style-type:disc;">Press together and close container</li><li style = "counter-reset:ol0;list-style-type:disc;">Place in blotting apparatus and add lotting buffer</li><li style = "counter-reset:ol0;list-style-type:disc;">Run for 1h at 100V 350-500mA</li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Fill a tray with blotting buffer</li><li style = "counter-reset:ol0;list-style-type:disc;">Transfer SDS gel to the blotting buffer</li><li style = "counter-reset:ol0;list-style-type:disc;">Assemble blotting sandwich (take it, soak sponge and place on it, soak filter paper and place on it, place inverted SDS gel on top, without creating bubbles.Add charged PDVF membrane with forceps ( charged in 100% ethanol and then soaked in buffer ) </li><li style = "counter-reset:ol0;list-style-type:disc;">Add soaked filter </li><li style = "counter-reset:ol0;list-style-type:disc;">Streak out bubbles</li><li style = "counter-reset:ol0;list-style-type:disc;">Add soaked sponge</li><li style = "counter-reset:ol0;list-style-type:disc;">Press together and close container</li><li style = "counter-reset:ol0;list-style-type:disc;">Place in blotting apparatus and add lotting buffer</li><li style = "counter-reset:ol0;list-style-type:disc;">Run for 1h at 100V 350-500mA</li></ul></div></div>

[STEPS]
TITLE: Microbiome and eDNA sampling of sediment
AUTHORS: Daniel S Read

[DESCRIPTION]
Protocol for field based sampling of bacterial biomass or environmental DNA (eDNA) in freshwater sediment.

[STEPS]
SECTION: Sample collection
1. To collect the sediment sample, you will require a sampling pole with a 50 mL falcon tube slot on the end, a 50 mL falcon tube, and a pair of gloves.
SECTION: Sample collection
3. Attach the falcon tube to the end of the sampling pole by placing it in the slot, and make sure it is secure.
SECTION: Sample collection
5. Use the open falcon tube at the end of the pole to scoop up the sediment by dragging it through the sediment. You want as much sediment and as little water as possible. You may need to drag the pole/tube several times.
SECTION: Sample collection
6. Once the sediment is in the falcon tube, carefully lift the pole and remove the tube from it. Replace the lid back on the falcon tube and ensure it is secure.
SECTION: Sample collection
2. Put the gloves on and unscrew the lid of the 50 mL falcon tube. Ensure you keep the lid of the tube in a safe place while you are taking the sample.

SECTION: Sample collection
4. Carefully walk to the river, and once in the area of the river where you would like to take the sediment sample, reach the pole with the falcon tube inside down to the river base.
 

SECTION: Sample collection
8. Store the sediment in the 50 mL falcon tube at -20 °C , or at the very least keep at 4 °C and out of direct sunlight, until returning to the laboratory. Once in the laboratory, store at -20 °C until analysis.

SECTION: Sample collection
7. Optional preservation buffer step - if you do not require this step, skip to Step 8.

If you would like to add a preservative buffer to the sediment, put a small amount (less than 0.5 g) into a 2 mL sterile Eppendorf microcentrifuge tube and add 1 mL of Zymo DNA/RNA shield to the tube. Shake the tube to thoroughly mix the sample with the buffer. The sample will then be relatively stable at room temperature for days-weeks, however, it is best to get the sample into cold storage, or preferably frozen at -20 °C as soon as possible once returning to the laboratory. Step 8 is not required if you have followed Step 7, unless you require extra samples to be collected or want spares/backups.
TITLE: K1 Keto Pills | Is It Really Fake Or Trusted?More Useful Than Other Products!
AUTHORS: thomsalonzo  

[DESCRIPTION]
 
      K1 keto   Life advanced weight loss true ingredients to help you get into ketosis   without having to go on a strict ketogenic diet for days.    (Buy Now)=>>   https://reviewbylogix.online/k5an  
 

[STEPS]
TITLE: ISOLATING CELLS IN LOUPE BROWSER
AUTHORS: Sumeet Gupta

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:center"><span style = "font-weight:bold;:UNDERLINE;">ISOLATING CELLS IN LOUPE BROWSER</span></div></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describes the steps used to analyze shotgun sequences deposited in in Integrated Microbial Genomes and Microbiomes (IMG/M ) by using functional anotation evidences. KEGG orthology terms and pathways  are used as evidence.The abundance of each of the corresponding biomarker genes is estimated by calculating the proportion of amino acid sequences assigned to the KO of interest (estimated gene copies, assembled and unassembled metagenomes, as retrieved from IMG/M) and normalized with respect to the total number of sequences assigned to KOs in each metagenome.  

[STEPS]
SECTION: Find metagenomes in the database
?. 
SECTION: Normalize the KO table
?. 
SECTION: Get the KO table from the database
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides a standardized methodolgy for a single-nucleotide polymorphism (SNP) at position -173 (-173*G/C) of the MIF gene. It also includes a standarized methodology to amplify a 207 bp (CATT<sub>5</sub>) segment of the MIF promoter region (MIF-794*CATT<sub>5-8 </sub>genotyping (rs5844572)) containing the microsatellite repeat sequence.</p>

[STEPS]