eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Oral cancer is one of the most significant cancers in the world; the most common of which is oral squamous cell carcinoma (OSCC) that makes up to 94% of oral malignances (Bernestein et al, 2013). Based on reports of the world Health Organization (WHO), OSCC occupied the sixth most common cancer in the world, 
Despite the advances and extensive research on novel approaches, current treatments are still limited to surgery, radiotherapy and conventional chemotherapy (Rosenthal et al, 2016). To date, chemotherapy is one of the most effective treatments (Wang et al, 2015). Among the most important anti-neoplastic drugs, anthracycline antibiotic doxorubicin (DOX) is commonly used in cancer therapy
In the past few years, the research field of nanomedicine has rapidly developed great promises in fighting cancer, and held the hope of providing new selective methods which can destroy cancer cells without harming healthy tissues (Zardini et al, 2015). Silica nanoparticles are among the most promising organic nano-biomaterials which were approved by the US Food and Drug Adminstration (FDA) for cancer therapy clinical trials (Wang et al, 2018). They have many advantages including their tunable particle size ( from 50-300 nm) which facilitate endocytosis by living cells, in addition to large pore volume and surface area for better control over drug loading and release (Zhou et al, 2018).

[STEPS]
TITLE: Fecal sample collection
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Fecal sample collection from mice to use for gut microbiome sequencing analysis.

[STEPS]
1. Set up the experiment by arranging clean cages without food, water or bedding. It is advisable to label each cage to avoid confusion later on.
2. In front of each cage, place a glass filled with dry ice and a 1.5ml Eppendorf tube to
speed up collection. Use plastic forceps each cage. Avoid using the same forceps to avoid contamination between samples.
3. Place the mice in their respective cages.
4. Collect all fecal pellets produced, and place it on the tube on dry ice. Do not collect pellets that were in urine or dried up pellets.
5. Transfer quickly the tube into a -80 degree fridge until DNA extraction.
TITLE: 10x Protocols: Chromium Next GEM Single Cell 5&#39; -- University of Minnesota TMCsTMCs (CG000331 Rev E)
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
 Following single-cell dissociation or nuclei isolation, the Next GEM 5' assay uses microfluidics to partition and assign cell- or nuclei-specific barcodes to transcript cDNA at the 5' end. Barcoded cDNA is prepared with adaptors for sequencing by synthesis (SBS). The following protocol has been used at the University of Minnesota TMCs in collaboration with the University of Minnesota Genomics Center. The following protocol has been adapted from protocols developed by 10x Genomics and Illumina to be used at the University of Minnesota TMCs in collaboration with the University of Minnesota Genomics Center. These protocols are owned by their respective companies and are subject to periodic revision.

[STEPS]
SECTION: Library Preparation
2.   

 


 

SECTION: FASTQ Generation
3. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq version 2.20.0
SECTION: Tissue Preparation
1. Complete single cell or nuclei isolation prior to starting this protocol
TITLE: 0.1% BSA media prep and flow
AUTHORS: Olya Oppenheim

[DESCRIPTION]
Fetal Bovine Serum contains traces of ligands of the BMP pathway which affect the flow experiments with the ibidi perfusion system.
replacing EGM2 with 2% with EGM2 with 0.1% BSA 4h prior to onset of flow (with same media) allows cells to respond better and collectively to flow.

[STEPS]
SECTION: BSA 0.1% media prep
1. 50 mL EGM2 without FBS
667 µL BSA 7.5% solution
filtrate through 0.22um yellow filter.
Add to slides 4h before starting flow.
SECTION: Optional
2. adding BMP9/10 to flow experiment
in order to obtain a final 0.5 ng/ml , add 1.4ul BMP9/10 of 10 ug/ml initial concentration per perfusion unit (volume of 14ml)
add this to each perfusion unit during slide connection
TITLE: SOP for Flow cytometry after DSS-induced gut and brain injury
AUTHORS: Malu G Tansey

[DESCRIPTION]
SOP for Flow cytometry after DSS-induced gut and brain injury

[STEPS]
1. Following isolation of brain and circulating immune cells with magnetic beads, cells were transferred to a V-bottom plate for surface epitope labeling. 
2. Samples were centrifuged at 300g for 5min at 4°C, washed once in PBS, re-pelleted, and resuspended in an antibody cocktail containing fluorophore-conjugated antibodies and LIVE/DEAD Fixable Blue stain (ThermoFisher, #L34962) in BD Horizon Brilliant Stain Buffer (BD Biosciences, #563794). 
3. Cells were incubated with antibodies for 20min at 4°C in the dark before three washes in FACS buffer containing 0.25mM EDTA, 0.01% NaN3, and 0.1% BSA in PBS. 
4. Labeled cells were fixed in 1% paraformaldehyde in a phosphate buffer for 30min at 4°C in the dark. 
5. Cells were washed three times in FACS buffer and transferred to FACS tubes in FACS buffer for flow cytometry. 
6. For compensation, 1uL of antibody was added to reactive and negative AbC Total Antibody Compensation Beads (ThermoFisher, #A10497) and 0.5uL of LIVE/DEAD Fixable Blue stain was added to ArC Amine Reactive Compensation Beads (ThermoFisher, #A10346). 
7. Bead staining occurred at the same time as cell staining and was subjected to the same protocol described above without fixation (e.g., labeled beads remained in FACS buffer while cells were in
fixative). 
8.  Samples were run on a BD FACSymphony A3,capturing either 100,000 single cells or the entire sample. Laser voltages were set with the assistance of SPHERO Supra Rainbow particles (Spherotech, Inc., #SRCP-35-2A) based on settings optimized during the development of this antibody panel. Compensation controls were calculated to ensure successful channel compensation prior to running samples. Raw files were analyzed using FlowJo (BD Biosciences) v.10.9.0 and gates were drawn with the assistance of fluorescence-minus-one controls.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is an effective protocol for genotyping diatom exconjugants which we believe is readily applicable to any other genetically manipulated marine microeukaryote.</p>

[BEFORE_START]
<p>1. Your exconjugant colonies should be growing as small - ~300 µL - liquid cultures in the presence of appropriate antibiotics. A 48-well plate works great for this purpose.</p>
<p> </p>
<p>2. Adjust primer Tm using <a href="https://www.thermofisher.com/us/en/home/brands/thermo-scientific/molecular-biology/molecular-biology-learning-center/molecular-biology-resource-library/thermo-scientific-web-tools/tm-calculator.html" target="_blank">this Tm Calculator</a>. </p>

[GUIDELINES]
<p>1. We find <a href="https://www.thermofisher.com/order/catalog/product/F160L" target="_blank">Phire Plant Direct PCR Master Mix </a> to be a top choice for direct genotyping of diatom exconjugants. </p>
<p> </p>
<p>2. Review protocol details <a href="https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0012959_Phire_Plant_Direct_PCR_MasterMixes_UG.pdf" target="_blank">here</a>.</p>
<p> </p>
<p>3. Note that we regularly run 10 µL reactions as opposed to 20 µL which is the minumum volume recommended by the manufacturer. </p>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Nutrient solution for rice hydroponics culture
AUTHORS: John Platten

[DESCRIPTION]
This represents a heavily modified Yoshida's solution.  Compared to Yoshida's formulation, this solution has almost the same concentration of macro and micronutrients, but the formulation has been simplified.  Key advantages:
* The solution is now formulated as three solutions at 1000× concentration, greatly simplifying dilution.
* The solution is less acidic than Yoshida's formulation, reducing the amount of hydroxide required to adjust pH.
* The solution eliminates Na+ and almost all Cl- from the medium, allowing independent investigation of toxicities of these ions.  In addition much of the K+ is supplied as KOH, potentially allowing control of the Na/K ratio independently of absolute concentrations and of Cl- levels.


[STEPS]
SECTION: Solution A, 1000× (Main macro and micronutrients)
1. Make this solution up as a SINGLE stock solution.  For 1L of stock solution,
* Start with ~600mL de-ionised water.
* Weigh out and dissolve each component (except ferric chloride and citric acid) directly in the 600mL solution.  Allow each to dissolve completely before adding the next.
* For the KOH, add pellets slowly, a few at a time, with constant mixing.
* Dissolve the citric acid separately in 100mL de-ionised water.  Dissolve the ferric chloride 6-hydrate directly in the citric acid solution.  Stir the ferric chloride-citrate solution for 15 minutes, then add slowly to the main stock solution while stirring.
* Make up to 1L.
SECTION: Solution B, 1000× (Ca, NO3)
2. For 1L of stock solution, dissolve the calcium nitrate 4-hydrate directly in 700mL de-ionised water.  Make up to 1L.
SECTION: Solution C, 1000× (Mg, S)
3. For 1L of stock solution, dissolve the magnesium sulfate 7-hydrate in 700mL de-ionised water.  Make up to 1L.
SECTION: Solution D, 1000× (silicon)
4. (Optional): For 1L of stock solution, dissolve 4.50g sodium metasilicate 9-hydrate in 700mL de-ionised water.  Make up to 1L.
The sodium metasilicate can also be weighed out and dissolved directly in the growth medium at the time of preparation.
SECTION: Preparation of hydroponics medium
5. For 100L of 1× hydroponics solution:
* Prepare ~90L of water.
* Mix in 100mL of each stock solution, mixing well before adding next stock solution.
* Add sodium metasilicate 9-hydrate (0.00450g/L) and dissolve with stirring, if not done as solution D.
* Adjust pH to 5.5
* Dispense to growth trays.
SECTION: Preparation of hydroponics medium
6. Monitor and adjust pH of stock solution daily at pH 5.5 with KOH/HCl as required.
Refresh stock solution twice per week.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Caenorhabditis elegans, a small (adults are ~1 mm long), free-living soil nematode that feeds on bacteria, is an ideal organism for applying various live microscopy techniques. The organism is transparent, thus it is possible to microscopically analyze and record the whole animal throughout its entire life. Its complete cell lineage is known, making it possible to follow developmental and differentiation processes in real time.

[STEPS]
SECTION: Pre-Imaging set-up
?. 
SECTION: Actual Imaging
?. 
SECTION: Post Imaging
?.
TITLE: Solid fungal extraction and C8 reversed phase chromatography
AUTHORS: Shara Van De Pas, Siouxsie Wiles

[DESCRIPTION]
Protocol for freeze-drying fungal cultures and preparing extracts using C8 reversed-phase chromatography.

[STEPS]
SECTION: Crude extraction of fungal compounds
1. Subculture fungus onto ~40 Petri dishes of media of choice and seal the plates with parafilm. Grow at the appropriate growth temperature until the fungus reaches the required age or coverage. 
SECTION: Crude extraction of fungal compounds
4. Weigh a clean 500ml beaker and break up dry fungal tissue into small pieces. Reweigh the beaker to obtain the dry weight of the fungus.
SECTION: C8 reversed phase chromatograpahy
11. Dissolve the remaining crude product in 10%M MeOH before loading onto C8 reversed-phase silica gel. Elute with a gradient of H20:MeOH (25% increments of 100ml volume) to afford five fractions (F1-F5). Apply pump pressure and collect the fractions in 250ml round bottom flasks. 

  

SECTION: C8 reversed phase chromatograpahy
12. Dry the 5 fractions at reduced pressure on the rotary evaporator at 130mbar (MeOH) and 40mbar (Water).
SECTION: NMR and Samples for Biotesting
13. When flasks are dry add 0.35-0.4ml of D4 MeOH to each flask (Fraction 1 isn't necessary as it will contain sugars and salts) and mix. Using a 1ml syringe and needle draw up the methanol-fungi mixture into the syringe and add it to a clean, labelled NMR tube.
SECTION: NMR and Samples for Biotesting
14. Cap samples gently and load them on to the AvanceIII-400mhz instrument. 
Name each of the samples and use these settings:
Proton H+
64 scans
Solvent: d4 Methanol
SECTION: NMR and Samples for Biotesting
15. Label and weigh 5 empty biotesting vials for further testing. Add the contents of the NMR tubes to each of the tubes and allow to dry. Reweigh the dry tubes and label them with the mass (in milligrams). Parafilm the lids and send back for extract testing.
SECTION: Crude extraction of fungal compounds
2. Once grown, freeze plates overnight  (-20 °C)
SECTION: Crude extraction of fungal compounds
3. Freeze dry over two days (-80 °C) until plates are dry and crisp.
SECTION: Crude extraction of fungal compounds
5. Submerge dry fungal tissue in methanol (MeOH) for 4 hours.
SECTION: Crude extraction of fungal compounds
6. Filter into a 500ml round bottom flask.
SECTION: Crude extraction of fungal compounds
7. Dry at reduced pressure on a rotary evaporator at 130 mbar. 
SECTION: Crude extraction of fungal compounds
8. Re-submerge fungal tissue in dichloromethane (CH2Cl2), cover with tinfoil and leave to soak overnight. 
SECTION: Crude extraction of fungal compounds
9. The following day filter into the same round bottom flask and dry at 500 mbar.
SECTION: C8 reversed phase chromatograpahy
10. Remove some crude product from the flask for downstream biological testing.
TITLE: Soil extract for cocultures host-parasites media
AUTHORS: Roscoff Culture Collection, Estelle Bigeard, Laure Guillou

[DESCRIPTION]
How to prepare soil extract



[BEFORE_START]
Please refer to our general recommendations to grow cultures :

https://www.protocols.io/private/A48906DC1374AD6281495CB86A8F092F

[STEPS]
SECTION: Preparation of the soil extract solution (25g/l)
3. Add 1200ml of MilliQ water to the soil. 
Close the erlenmeyer with a aluminium foil.
 
SECTION: Preparation of the soil extract solution (25g/l)
4. Heat at 100°C with pemanent mixing during 1 hour to obtain an efficient boiling.
Adjust this time depending on the volume (more volume, more time).
Let cool.
 
SECTION: Preparation of the soil extract solution (25g/l)
5. Filtrate on 0.2 µm, using a glass prefilter and a pump vacuum. 
As the prefilter will be rapidly clogged, better to add the boiled soil 100mL per 100mL.
If this happens, replace the glass prefilter, then the filter.
Transfer the solution in one or several glass bottles.
SECTION: Preparation of the soil extract solution (25g/l)
7. The day after, when the medium is at room temperature, place it at 37°C for 24 to 48 hours.
Then autoclave again at 121°C for 20 minutes.

This step allows to reactivate spores (fungi etc) contained in soil which have not been destroyed by the first sterilization, and to kill them during the second one. 
 
SECTION: Preparation of the soil extract solution (25g/l)
8. Add 50ml of soil extract per 1 L of medium.
SECTION: Soil sampling
11. Soil is sampled in a pine wood near Roscoff, Brittany, France. 
Each sampling is placed in plastic boxes.
Back to the laboratory, plastic boxes are placed at 37°C for 12 to 24 hours to dry it.
SECTION: Preparation of the soil extract solution (25g/l)
2. Weigh 30g of dried soil and place it in a 4L glass erlenmeyer.
SECTION: Soil sampling
1. Soil is sampled in a pine wood, located in Brittany, France.

To find a correct sampling area in your country, try to find a location devoid of pesticides and pollutants. It should be also a place where you know that you can collect the soil again and later in order to always use the same soil, especially if this one worked.

Collect the soil in plastic boxes. 
Back to the laboratory, plastic boxes are placed at 37°C for 12 to 24 hours to let the soil dry. The soil must be dry before use, otherwise the boiling step should be longer.
SECTION: Preparation of the soil extract solution (25g/l)
6. Autoclave at 121°C for 20 minutes.
Place the bottle of the filtered-boiled soil under a safety cabinet, and let it cool.
TITLE: Transforming E. coli
AUTHORS: Brian    Teague

[DESCRIPTION]
Transformation is the process of inducing chemically competent E. coli to take up a plasmid. We do this in order to use E. coli as a "DNA copier" -- the bacterium takes up the plasmid, then copies it as it copies its own genomic DNA. We can grow up as many E. coli as we like in a liquid culture -- each of which has 20-50 copies of the plasmid -- then extract the plasmid DNA back out of the bacteria in the culture.

  


[GUIDELINES]
Time and temperature are critical parameters here -- make sure to follow the instructions very closely. For example, if the instructions read "heat shock in a water bath at 42 °C for exactly 30 s", don't grab your tube out of the ice bucket, wander over to the water bath, get distracted chatting to friends, etc. Instead, bring your ice bucket with your cells to the water bath, put the cells in the water bath for 30 seconds, and then put them immediately back on ice.


[STEPS]
SECTION: Transformation
1. Retrieve the two tubes of chemically competent E. coli cells from the -80 °C freezer and place them immediately on ice. Incubate the cells on ice 3-4 minutes to thaw.

SECTION: Transformation
2. Add 1 µL of DNA from your ligation to a tube of cells. Immediately after adding the DNA to the tube, flick several times to mix, then immediately return to ice.

SECTION: Transformation
3. Add1 µL of DNA from the transformation control to a separate tube of cells. Immediately after adding the DNA to the tube, flick several times to mix, then immediately return to ice.

SECTION: Transformation
4. Incubate the cells on ice for 30 min

SECTION: Transformation
5. Heat shock the cells for exactly 30 s in the 42 °C  water bath.

 


SECTION: Transformation
6. Incubate the cells on ice for 2 min

SECTION: Transformation
7. Add 250 µL   to each tube.

SECTION: Transformation
8. Tape each tube to the platform of an incubating shaker and shake   

SECTION: Plating
9. Label the kanamycin selection plates appropriately.

 

 

SECTION: Plating
10. Shake ~10   onto each plate.

SECTION: Plating
11. For each transformation, pipette 100 µL onto the center of the plate.

SECTION: Plating
12. Cover the plates and shake the beads around to spread the cells out.
SECTION: Plating
13. Dispose of the beads by tapping them into the waste container.
SECTION: Plating
14. Incubate the plates upside down in an incubator, 37 °C 2 min
TITLE:  Preparation of a protein-AG conjugated to horseradish peroxidase by the periodate method.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A recombinant protein that combines the IgG-binding domains of SpA and SpG was developed, and labelled to horseradish peroxidase. It was used as universal conjugate in ELISA for the assessment of antibodies against Brucella spp in cattle, sheep, dogs, goats and pigs. It was reported that similar results as the one shown using the chimeric protein AG were obtained when murine monoclonal antibody-enzyme conjugates were used [1,2].    </div><div class = "text-block">References</div><div class = "text-block"><span>1. Justiz-Vaillant AA, Akpaka PE, McFarlane-Anderson N, Smikle MF. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.</span><span style = "font-style:italic;">West Indian Med J</span><span>. 2013;62(1):12-20.</span></div><div class = "text-block">2. Justiz-Vaillant AA, McFarlane-Anderson N, and Smikle M. “Bacterial Immunoglobulin (Ig)-Receptors: Past and Present Perspectives.” American Journal of Microbiological Research, vol. 5, no. 2 (2017): 44-50. doi: 10.12691/ajmr-5-2-4.</div></div>

[STEPS]
?. Horseradish peroxidase (500 µg in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml) followed by incubation in the dark for 2 h.
?. Mix 500 µg of staphylococcal protein-A (SpA) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate. On the other hand mix 500 µg of streptococcal protein-G (SpG) with an equal amount (500 micrograms) of the mix of horseradish peroxidase-sodium periodate.
?. The two mixtures are incubated separately for 3 hours at 4°C with gentle agitation.
?. Forty µl of freshly prepared NaBH4 solution (5 mg NaBH4 /ml 0.1 mM NaOH) is then added separately  to the preparations, which are centrifuge (13,000rpm., 10 minutes at RT). Add to each preparation cold saturated amonium sulphate solution and centrifuge again (10000rpm, 25 minutes at 4°C).
?. Now mix the SpA-HRP preparation with SpG-HRP and incubate the mixture for 90 min at 4°C in the dark with gentle agitation.
?. The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
?. The pellet (SpAG-HRP) is re-suspended in 500 µl of PBS pH=7.4 and dialysed against 1L of PBS for 24 h with 3 buffer changes.
?. An equal volume of glycerol is added to the dialysate followed by 200 µl of bovine serum albumin, BSA (20 mg/ ml).
?. The SpAG-HRP conjugate is then stored at -20°C until further used.
TITLE: Vivarium Population Spender: Immigration Module
AUTHORS: Camila Rangel Smith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Description of the steps followed by Vivarium Population Spenser library when running the Immigration module. </div></div>

[STEPS]
?. Get the total number of immigrants expected for that local authority on a year from a local authority level international migration table.
?. Divide the number of total immigrants to be assigned by the number of steps existing in that year.
?. For each time step:
?. Add the fraction of immigrants to be assigned in one time step to the state table of that local authority.
?. Assign the new immigrants an MSOA based on their age and gender from rates extracted from table of international migration at the MSOA level.
?. Assign these new immigrants an ethnicity, age and sex sampling from rates extracted from the local authority level international migration table.
TITLE: Glycerol stock preparation 
AUTHORS: Ashwinuday  

[DESCRIPTION]
This protocol is for the preparation of 10% glycerol stocks of bacterial strains.

[STEPS]
1. From a streaked plate of the bacteria, pick one colony and inoculate into a test tube containing 10 mL  1X LB
2. Grow the culture till it reaches an OD ~ 0.6
3. Into cryo-vials add 1.6 mL culture and 0.4 mL 50% autoclaved glycerol.

 

4. Flash freeze the tubes in liquid Nitrogen and store it at -80 °C