eamag/protocols-with-steps huggingface dataset:
TITLE: Vivarium Population Spenser: Internal migration module
AUTHORS: Camila Rangel Smith, Kasra Hosseini

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

[STEPS]
?. This module models  the [internal_migration](src/vivarium_population_spenser/internal_migration.py) between MSOAs (and theirrespective LADs) of individuals based on their gender, age, initial location (local authority level) and ethnicity.The input table to establish the pool of migrants that internally migrate based on  their characteristics is [InternalOutmig2011_LEEDS2.csv](persistant_data/InternalOutmig2011_LEEDS2.csv). Once the pool of migrants is chosen they are assigned to a new MSOA based on their age and gender using the MSOA migration matrices in [od_matrices](persistant_data/od_matrices).
?. Divide the annual outmigration rates for that local authority by the number of time steps existing in a year.
?. For each time step:
?. Select all individuals in the sample that appear as "alive", have an associated gender and haven't internally migrated in the las 365 days.
?. For these individuals, get the internal outmigration rate given their age, gender, ethnicity and location and turn it into a probability.
?. Using random sampling and the internal outmigration probability of the individuals, choose which individuals are going to internally migrate in that time step.
?. From this pool of individuals use an age and gender specific MSOA to MSOA migration matrix to choose which MSOA (and their respective local authority)  they are going to migrate to.
?. Update their information in the state table with their new location and the time they internally migrated.
TITLE:       ELISA for quantification of IL-34 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>Various phagocytes and epithelial cells synthesize Interleukin-34 (IL-34). It enhances IL-6 production and participates in the differentiation and development of antigen-presenting cells including microglia. </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-34 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-34 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-human IL-34 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-34 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) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-34 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 IL-34 standard dilutions and the human IL-34 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Generation of stable cell lines using retroviral system
AUTHORS: nguyen.tha

[DESCRIPTION]
This protocol details generation of stable cell lines using retroviral system.

[GUIDELINES]
Attention

The HEK293T cells detach very easily, be extra gentle when changing the media.


[STEPS]
SECTION: Day 1
1. Seed NIH HEK293T cells into a 6-well plate (900k cells/well if set up in the morning, 950k cells/well if set up in the afternoon). 
 

SECTION: Day 2: The following protocol is designed for one well of the 6-well plate
2. Transfect cells with viral and helper vectors using lipofectamine LTX. Combine the following in a 1.5 mL tube:
 
      viral vector   construct (pBMN, pBABE or pMX)  1.5 µg    well gag-pol vector  1.0 µg    well VSV-G vector  0.5 µg    Opti-MEM (RT)  500 µL  
 
SECTION: Day 2: The following protocol is designed for one well of the 6-well plate
3. Add 3 µL of Plus reagent and mix well. Incubate at Room temperature for 5 min.
SECTION: Day 2: The following protocol is designed for one well of the 6-well plate
4. Add 9 µL of Lipofectamin LTX (1:3 ratio of Plus:LTX is standard in the lab but can be adjusted for your own protocol) and vortex for 15 s. Incubate at Room temperature for 20 min.

SECTION: Day 2: The following protocol is designed for one well of the 6-well plate
5. Once the 20 min incubation starts, replace the media in each well with 1 mL DMEM/10% FBS media.
SECTION: Day 2: The following protocol is designed for one well of the 6-well plate
6. When the 20 min incubation finishes, add the optimum/liposome mix to the well. 
 

SECTION: Day 3
7. In the morning, remove the old media from the HEK293T cells which may contain viruses at this stage) into a beaker of beach and add 1 mL of fresh growth media. The next day, viruses can be harvested for infection.

SECTION: Day 3
8. Seed the target cells (about 100k-120k cells) into a 6-well plate if intending to do infection with fresh viruses. 
SECTION: Day 4
9. In the late afternoon, collect viral supernatant from HEK293Ts, spin down at max speed for 5 min to pellet debris and filter through 0.45µm syringe filters. Viral particles can freshly be used for infection on the cells plated out on day 3 (see below) or can be frozen at -80 °C for future use.

SECTION: Day 4
10. For second harvest, add 1.5 mL fresh growth media back to HEK293T cells for 2 days and harvest again (on Day 6).
SECTION: Day 4
11. For infection, harvested viruses are topped up with fresh growth media to make up a total of 2 mL.
SECTION: Day 4
12. Aspirate the media from the target cells.
SECTION: Day 4
13. Add the 2 mL of virus-containing media (from step 3) to the target cells. Add polybrene to a final concentration of 8 µg/mL to the well and mix well.
SECTION: Days 5 and 6
14. The viruses can be removed from the cells into a beaker of bleach after 24 h (Day 5) or 48 h (Day 6) and fresh media can be added to the wells.
SECTION: Days 5 and 6
15. All waste must be treated as viral waste for at least 3 media changes over 3 days post-infection.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Current long read sequencing, e. g. PacBio and Nanopore, requires high molecular weight (HMW) and highly pure DNA. Many fungal and plant species have high content of polysaccharides and other contaminants that are co-precipitated with DNA during ethanol precipitation. This protocol aims to purify HMW DNA from the DNA solution obtained using Benjamin Schwessinger's protocol (dx.doi.org/10.17504/protocols.io.ewtbfen).</p>
<p>The protocol was tested fuccessfully in wheat leaf rust, barley leaf rust, wheat stem rust and myrtle rust.</p>
<p>Critcal step to seperate HMW DNA from low molecular weight DNA and other contaminants during ethanol precipitation is to allow cotton-fiber-like HMW DNA to sediment by gravity, while other small molecule DNA and contaminants remain in the supernatant.</p>

[BEFORE_START]
<p><strong>Reagents required:</strong></p>
<p>Pectinase (Sigma P2611)</p>
<p>RNAse A (QIAGEN 19101)</p>
<p>Proteinase K (NEB P8172)</p>
<p>Buffered Phenol:Chlororform:Isoamylalcohol (25:24:1, Sigma P2069)</p>
<p>3 M Sodium Acetate pH 5.2</p>
<p>Isopropanol</p>
<p>70% ethanol</p>

[GUIDELINES]
<p>All steps need gentle pipetting, no vortex.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Successful and cost-effective DNA extraction  method from insects with small amount of tissue 
AUTHORS: Rahul Jamdade

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">DNA extraction from insects is an initial and critical  step that might affect the molecular taxonomy. There are several protocols for efficient DNA extraction published by researchers, which might be helpful for others working in the relevant field. There are also some  commercial kits optimized to extract DNA even from low volumes or less amount of tissue. Research has conventionally showed that Phenol:  chloroform: Isoamyl alcohol (PCI) method gives good yield and is cost effective as compared to other methodologies, especially commercial kits. Hereby is the CTAB and  PCI protocol, which gives good yield from insect tissue preferably legs,  head and wings (avoid using whole body).</div></div>

[STEPS]
TITLE: Sucrose preference test 
AUTHORS: mariangela.massarocenere

[DESCRIPTION]
The sucrose preference test assesses the animal’s interest in a sweet-tasting sucrose solution relative to unsweetened
water

[STEPS]
1. Place one animalper cage during the test duration and habituate them to drinking from two equally accessible bottles 
3. Refill each bottle with fresh water or 1% sucrose solution and inverte to its original position every day for two consecutive days 
2.1. To avoid side preference, place the sucrose bottle on the left for half of the cages and on the right for the other cages
2. Expose animal to a double-choice drinking test, with the two bottles containing one sucrose solution (1%) and the other drinking water
4. After 72h, measure with a graduated cylinder and record water and sucrose solution consumption 
5. Calculate the percentage of sucrose intake and water intake as follows:
       and
TITLE: Flow CyTOF Using Single Cells From Human Islets
AUTHORS: Klaus H. Kaestner Lab, Suzanne Shapira

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Cytometry by time-of-flight (CyTOF) is an application of mass cytometry using antibodies conjugated to rare heavy metal isotopes combined with mass spectrometry. This technique significantly reduces background signals and spectral overlap often seen with traditional fluorescence methods. This protocol describes a technique to analyze multiple cellular features of human islets in parallel using a defined and optimized antibody cocktail.</span></div><br/><br/><br/></div>

[STEPS]
?. [II. CyTOF barcoding and labelling]
Thaw the cells in  quickly, and immediately add the Perm bufferRemove the barcodes (Fluidigm 201060) from  freezer and allow them to warm up to RT for at least 10 min.
37 °C
-20 °C
?. [II. CyTOF barcoding and labelling]
Wash cells 2x with Foxp3 perm buffer (eBioscience, 00-5523-00), 700 xg (2900 rpm), 5min. Note: centrifuge at higher speed after fixation.
?. [II. CyTOF barcoding and labelling]
Resuspend each sample to be barcoded in  of perm buffer (aiming at 1 ~ 3x106 cells)
800 µl
?. [II. CyTOF barcoding and labelling]
Resuspend barcodes completely in  of perm buffer and transfer them to the appropriate samples. Mix the samples immediately and completely (pipette immediately).
100 µl
?. [II. CyTOF barcoding and labelling]
Incubate for 30 min at RT.
?. [II. CyTOF barcoding and labelling]
Centrifuge cells, wash twice with  of perm buffer. 700 xg. 5 min.
2 mL
?. [II. CyTOF barcoding and labelling]
Intracellular stain with master mix in perm +1% FBS at  overnight. Master mix is 100ul/million for each tube.Master Mix:
4 °C
?. [II. CyTOF barcoding and labelling]
Wash with 2x perm buffer at 700 xg, 5min.
?. [II. CyTOF barcoding and labelling]
Wash 2x with PBS (700 xg, 5min)
?. [II. CyTOF barcoding and labelling]
DNA intercalator Iridium (FLuidigm 201192B, aliquoted in pcr tubes) at 1:4000 at RT for 1h in buffer to stain DNA is 2% PFA in PBS with Iridium.
?. [II. CyTOF barcoding and labelling]
Right before running samples on CyTOF2, wash (2x) with MilliQ H2O (700 xg, 5min)
?. [II. CyTOF barcoding and labelling]
Resuspend in MilliQ H2O. Adjust cell concentration to about 5x105/ml (expect lose 40% of the starting cells at this step)
?. [II. CyTOF barcoding and labelling]
Analyze on CyTOF at about 300-500 evt/sec
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Materials:</strong><br /><br />
<ol>
<li>Reagent “A” - 2.0% Na<sub>2</sub>CO<sub>3</sub> in 0.1 N NaOH</li>
<li>Reagent “B” - 0.5% CuSO<sub>4</sub><sup>.</sup>5H<sub>2</sub>O in 1.0% Na citrate</li>
<li>Reagent “C” - 50.0 mL reagent “A” + 1.0 mL reagent “B”</li>
<li>Reagent “D” - Folin-Phenol Reagent (2 N Solution, Folin/Ciocalteau), dilute 1:1 with d-H<sub>2</sub>O before using</li>
</ol>
<strong><br />Notes</strong>:<br /><br />It may be possible to reduce the initial volume of each assay to 0.5 mL and thus reduce the amount of subsequent reagents used by 50%.  Use the same amounts of BSA for the protein standard curve.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Removal of Melanin
AUTHORS: Jason Stajich

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Principle is that CTAB is charging the anionic nucleotides whereby neutral polysaccharides/ melanins are remaining in supernatant. This method also uses urea with the idea that the presence of urea helps to solubilize hydrophobic compounds that would otherwise potentially interact with the hydrophobic core of the CTAB micelles.</div></div>

[STEPS]
?. Add to ~-  DNA/RNA solution until a volume of  is reached.
100 µl
200 µl
400 µl
?. Add
130 µl
?. Add mL of CTAB-Urea solution
1.6 mL
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?. Mix samples (by hand).
?. Incubate overnight at
4 °C
?. Centrifuge for  at max speed at
4 °C
?. Remove the solution. Be very careful in this step!
?. Resuspended in  µL
400 µl
?. Add 2  Vol of EtOH (100%)
[VOLUMES]
?. Incubate on ice for
?. Centrifuge: 15300 34, 15 min, 4 10
?. Wash with
?. Wash again with
?. Centrifuge: 20800 34, 10 min
?. Remove the supernatant.
?. Dry pellet
?. Resuspend in TE buffer
TITLE: Human fibroblast culturing
AUTHORS: Laura  Smith, David  C

[DESCRIPTION]
Fibroblasts are cultured in Dulbecco’s modified eagle media (DMEM) 4500 (mg/L) growth medium supplemented with Glutamax (Gibco), 10% foetal bovine serum (FBS), non-essential amino acids (NEAA: 0.1 mM of: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine) and penicillin/streptomycin antibiotic cocktail (50ng/ml) at 37°C and 5% CO2. 

[STEPS]
SECTION: Characteristics
1. Characteristics
Obtained /developed from human adult dermal biopsies
Proliferative until p20
Doubling time ~ 24h
Maintain between 60 – 90% confluent
SECTION: Complete growth medium
2. DMEM (LT      # 61965-059; 4500 mg/L and no pyruvate) Glutamax medium
Fetal      bovine serum (FBS, 10% final) – 1 / 10 dilution
Pen / Strep     (LT # 15140-122, 50 U/ml and 50 ng/ml final) -- 1      / 200 dilution
MEM / NEAA     (LT # 11140-035) -- 1 / 100 dilution
As      required -- Sodium pyruvate (Sigma #      S8636, 1 mM final) – 1 / 100 dilution
As      required -- Uridine (Sigma # U3003,      stock of 25 mg/ml in H2O filter-sterilised; 50 ug/ml final) – 1 / 500 dilution
As      required, as permitted by DSO -- Fungizone     (LT # 15290-026; original of 250 ug/ml) – 1 /      100 dilution
SECTION: Subculturing by trypsinisation, and cell counting
3. Wash cells      grown onto 10 cm Ø plate with PBS.
Add 1.5      ml diluted trypsin (4 ml of 2.5%, LT #      15090-046, into 100 ml Versene LT #      15040-033) and leave at 37’C for several mins.
Tap      the plates and add 2 ml complete medium to stop trypsination.
To a      50 ul sample, add equal vol of 0.4% Trypan blue (Sigma # T8154 or LT #      15250-061); fed it to a hemacytometer.
Optional      -- to pellet cells and resuspend into fresh medium.
Replate      the cells accordingly; typically 10 ml medium is used for 10 cm Ø plate.
Subcultivation      ratio of 1:4 is recommended; medium renewal every 2-3d.

SECTION: Freezing and thawing
4. 1.After centrifugation to pellet cells, resuspend in Freezing medium (90% FBS / 10% DMSO filter sterilised) and aliquot into freezing vials; typically ¼ of a 50% confluent 10 cm Ø plate of 125K cells in 0.5ml.Freeze cells in a stepwise fashion from -80’C to liq N.
2.Thaw cells at 37’C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Last optimized January 2006, updated by Matt on March 16 2009

[GUIDELINES]
This protocol comes from a group of other protocols.This protocol is (1) of (4): <br />1. <a href="https://www.protocols.io/view/Large-Volume-Marine-Cyanophage-Phage-Protocols-c3iykd" target="_blank">'Large Volume Marine Cyanophage Phage Protocols'</a><br />2. <a href="https://www.protocols.io/view/DNA-Extraction-Protocol-c32yqd" target="_blank">'DNA Extraction Protocol'</a><br />3. <a href="https://www.protocols.io/view/DNA-Precipitation-Protocol-c34yqv" target="_blank">'DNA Precipitation Protocol'</a><br />4. <a href="https://www.protocols.io/view/Checking-DNA-Concentration-with-Agarose-Gel-c35yq5" target="_blank">'Checking DNA Concentration with Agarose Gel'</a><br /><br />Needed:<br />
<ul>
<li>2L Pro99</li>
<li>Cyanophage stock</li>
<li>SYBR titering</li>
<li>10 mL chloroform / L phage lysate</li>
<li>Incubator</li>
<li>Centrifuge Beckman Ja-14 @ 12,500 rpm</li>
<li>Kim Wipes</li>
<li>Polyethylene Glycol (PEG)</li>
<li>Orbital shaker @ 150 rpm</li>
<li>CsCl step gradient (1.3, 1.4, 1.5, 1.65 bands)</li>
<li>Ultracentrifuge tubes</li>
<li>Concentrated phage samples</li>
<li>SW28 swinging bucket rotor @ 28,000 rpm</li>
<li>Slide-A-Lyzer dialysis cassettes</li>
<li>3M and 1.8M NaCo</li>
<li>NaCl buffer</li>
<li>Tris HCl</li>
<li>MgCl2</li>
</ul>
<br />RNase 100ng/ul = 100ug/ml ==&gt; 1:10 = 10ug/ml ==&gt; use 1 ml/L or use UD at 100 ul/L

[STEPS]
?. 
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TITLE:         ELISA for quantification of human C3 in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-human C3 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. Human C3 present in the serum or plasma 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-C3 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-C3 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) into each well.
?. Incubate the microwells in the dark for 20 min.
?. A colored product is formed in proportion to the quantity of C3 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 C3 standard dilutions and the human C3 sample concentration is determined.
TITLE: Practical Guide to Live Sampling of Livestock and Wildlife for Infectious Disease Surveillance
AUTHORS: Stefano Catalano

[DESCRIPTION]
Under- or misdiagnosed cases of disease caused by especially dangerous pathogens present public health and proliferation risks. Numerous studies have demonstrated that in low-resource settings, and particularly where malaria is endemic, patients presenting to healthcare facilities with febrile illness may not receive an accurate diagnosis due to, among other reasons, shortcomings in differential protocols and lack of available diagnostic tests. In Guinea, when patients present at the community or prefectural level with febrile illness, weaknesses in clinical differential protocols usually result in the patient being diagnosed with a common infection, notably malaria and occasionally typhoid, with no further testing performed. As such, acute febrile illness presents an unquantified and undifferentiated burden to the Guinean healthcare system. The specific challenge for public health authorities is that pathogens causing febrile episodes in the population, and the zoonotic transmission pathways that may drive them, have not been systematically nor comprehensively identified. Fever Project is a three-year research project with the objective to build capacity for threat reduction in Guinea and improve health security through an integrated approach to human and animal health for the identification of high-consequence aetiologies of acute febrile illness in humans. The project’s goals are to: I) identify the causative agents of acute febrile illness in humans in Guinea; II) uncover evidence for transmission of high-consequence zoonotic pathogens associated with acute febrile illness; III) investigate risk factors for acute febrile illness in humans, including exposure to domestic and peri-domestic animals; and IV) improve capacities for the detection of high-consequence pathogens associated with acute febrile illness.

[STEPS]
1.