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

[DESCRIPTION]
<p>A simple 96-well plate assay for stress resistance across a range of stress doses. Originally designed for yeast, but easily modifiable for any colony-forming microbe.</p>

[GUIDELINES]
<p>Stress assays are very sensitive to differences in growth conditions. In our hands, we have noticed that relatively small differences in temperature, shaking speed (aeration), and media composition can affect cellular stress resistance. To test media consistency when we receive a new batch of media, we perform control experiments on cells grown with both the old and new media lots. To maintain consistency across shaker platforms, we use a digital tachometer to ensure identical shaking speed, and we measure the temperature of a control flask of water every morning. </p>
<p> </p>
<p>Timing is also a very important consideration. We use multi-channel pipettors or pinners and work quickly, especially when transferring cells from the secondary stress to fresh media. We process a maximum of two 96-well plates at a time to ensure that viability differences across strains are not due to differences in exposure time to the secondary stress. This was determined in our lab empirically for our yeast strains using technical replicates across multiple plates, and we would recommend this procedure when adopting this protocol for new organisms or stress exposure times. </p>
<p> </p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Protocols for "Draft genome of the aquatic moss Fontinalis antipyretica (Fontinalaceae, Bryophyta)"
AUTHORS: Yang Liu, Huan Liu, Hongfeng Chen, Bernard Goffinet, Nikisha Patel, Ziqiang Chen, Shanshan Dong, Sibo Wang, Linzhou Li, Jin Yu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-style:italic;">Background:</span><span>Mosses compose one of the three lineages that form the sister group to extant vascular plants. Having emerged from an early split in the diversification of embryophytes, mosses may offer complementary insights into the evolution of traits following the transition to and colonization of land. Here, we report the draft nuclear genome of </span><span style = "font-style:italic;">Fontinalis antipyretica </span><span>(Fontinalaceae, Hypnales), a charismatic aquatic moss widespread in temperate regions of the Northern Hemisphere. Wesequenced and </span><span style = "font-style:italic;">de novo</span><span> assembled its genome using the 10 × genomics method. The genome comprises 385.2 Mbp, with a scaffold N50 of 45.8 Kbp. The assembly captured 87.2% of the 430 genes in the BUSCO viridiplantae odb10 dataset.The newly generated </span><span style = "font-style:italic;">F. antipyretica</span><span>genome is the third genome of mosses, and the second genome for a seedless aquatic plant.</span></div></div>

[STEPS]
TITLE: SARS-CoV-2 Milk ELISA
AUTHORS: Powell RL

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Currently, the COVID-19 diagnostics sector includes Point-of-Care and ELISA tests for IgG and IgM that can assess saliva or blood as described by the CDC [Centers for Disease Control and Prevention]. Similarly, the FDA webpage for EUA Authorized Serology Test Performance makes no reference to IgA COVID-19 assays [Food and Drug Administration]. Taken together, there are no direct competitors in the mucosal IgA quantitative diagnostics space.</div><div class = "text-block">To address this gap in diagnostic coverage we have generated a novel proof-of-concept dataset on the use of COVID-specific IgA antibodies as a marker of infection. As described below, our assay dataset demonstrates utility across several parameters:</div><div class = "text-block">-Highly specific</div><div class = "text-block">-Highly sensitive</div><div class = "text-block">-Works with undiluted human milk, simplifying sample collection and processing</div><div class = "text-block">-Defines a novel correlation between COVID-specific IgA and Secretory Component (SC), where SC is a known surrogate for dimeric IgA, the predominant IgA type in mucosal secretions</div><div class = "text-block">-Establishes that dimeric IgA antibodies detected by the assay are capable of neutralizing SARS-CoV-2</div></div>

[STEPS]
?. [Milk Processing]
Pour fresh milk into 50mL conical tube and centrifuge at 800g for 15min at room temperature
Centrifuge: 800 34, 15 min, 22 10
?. [Milk Processing]
remove fat using a sterile spatula
?. [Milk Processing]
carefully pour skim milk into a clean 50mL tube
?. [Milk Processing]
repeat centrifugation and fat removal 2x
?. [Day 1]
Coat half-area 96-well plates with 50ng SARS-CoV-2 spike protein in 50ul PBS
50 µl
?. [Day 1]
over and store overnight at 4C
4 °C
?. [Day 2]
Wash plates with 100ul 0.1% Tween 20/PBS (PBS-T) 6x
100 µl
?. [Day 2]
Block plates with 100ul 3% goat serum/0.5% milk powder/3.5% PBS-T for 1h at room temperature
100 µl
0 Room temperature
?. [Day 2]
Wash as above
?. [Day 2]
Add 50ul milk undiluted or diluted with 1%BSA/PBS to each well in duplicate
50 µl
?. [Day 2]
Incubate at room temperature for 2h
0 Room temperature
?. [Day 2]
Wash as above
?. [Day 2]
Add 50ul  horseradish peroxidase (HRP)-conjugated secondary antibody: goat anti-human-IgA at 1/3000 in 1%BSA/PBS.
50 µl
?. [Day 2]
Incubate at room temperature for 1h
0 Room temperature
?. [Day 2]
Wash as above
?. [Day 2]
Add 50u Pierce 1-Step Ultra TMB-ELISA Substrate Solution followed by 50ul 2N hydrochloric acid (HCl)
50 µl
?. [Day 2]
Measure OD at 450nm on an ELISA plate reader
TITLE: Grip strength test
AUTHORS: Marina Lorente Picón, Núria Peñuelas, Ariadna Laguna, Miquel Vila

[DESCRIPTION]
Grip strength test for mice

[STEPS]
1. Hold the animal by the middle/base of the tail and
allow it to grasp a tangled fine gauge stainless steel wire attached to steel
chain (13.2g). Then, lift them carrying the corresponding weight with
their forepaws for a total of 5 seconds. If the animal does not succeed assign 0
seconds to that animal. If the animal succeeds holding the weight, move on to the next weight.
2. Hold the animal by the middle/base of the tail and
allow it to grasp a tangled fine gauge stainless steel wire attached to steel
chain (32.1g). Then lift them carrying the corresponding weight with
their forepaws for a total of 5 seconds. If the animal does not succeed assign the max nº of seconds he held the weight to that
animal. If the animal succeeds holding the weight, move on to the next weight. 
3. Hold the animal by the middle/base of the tail and
allow it to grasp a tangled fine gauge stainless steel wire attached to steel
chain (19.7g). Then lift them carrying the corresponding weight with
their forepaws. If the animal does not succeed assign the max nº of secondsto that animal. If the animal succeeds holding the weight, move on to the next weight.
4. Hold the animal by the middle/base of the tail and
allow it to grasp a tangled fine gauge stainless steel wire attached to steel
chain (25.9g). Then lift them carrying the corresponding weight with
their forepaws. If the animal does not succeed assign the max nº of secondsto that animal. If the animal succeeds holding the weight, move on to the next weight.
5. Hold the animal by the middle/base of the tail and
allow it to grasp a tangled fine gauge stainless steel wire attached to steel
chain (38.4g). Then lift them carrying the corresponding weight with
their forepaws. If the animal does not succeed, assign the max nº of secondsto that animal. If they succeed holding the weight, move on to the next weight.
6. Hold the animal by the middle/base of the tail and
allow it to grasp a tangled fine gauge stainless steel wire attached to steel
chain (44.6g). Then lift them carrying the corresponding weight with
their forepaws. If the animal does not succeed, assign the max nº of secondsto that animal. If the animal succeeds holding the weight, assign 30 seconds to that animal.
7. Calculate Grip latency (s) as a sum of the time holding the increasing weights (0-30 seconds).
TITLE: Thawing MRC5 sub-stock
AUTHORS: Morrisey Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">MRC5 Media</span></div><div class = "text-block">450uls DMFM/F12 (Cell Center cat#11320033)</div><div class = "text-block">45uls 10% FBS (Invitrogen cat#10437-028)</div><div class = "text-block">5uls 1% pen/strep (Cell Center cat#15240096)</div></div>

[STEPS]
TITLE: Nuclear Extraction from Tissue for FACS
AUTHORS: anita.adami

[DESCRIPTION]
This protocol details the process for pre-tissue preparation and tissue preparation for nuclear extraction for FACS.

[BEFORE_START]
Keep everything On ice.

[STEPS]
SECTION: Pre-Tissue Preparation
1. Coat all tubes (centrifugation, sample and collection tubes) in coating medium   (1 % (v/v), filtered).

SECTION: Pre-Tissue Preparation
2. Cool ultracentrifuge to 4 °C prior to centrifugation

SECTION: Pre-Tissue Preparation
3. Pre-weigh the DTT needed.
SECTION: Pre-Tissue Preparation
4. Keep douncers on ice.

SECTION: Tissue Preparation
5. Put 2.8 mL in centrifuge tube.

SECTION: Tissue Preparation
6. Mix 6 mL  + 2 mL  in 15 ml falcon tube.

SECTION: Tissue Preparation
7. If the tissue is large (>2 mm3), chop the tissue with a razor.
SECTION: Tissue Preparation
8. Collect chopped tissue with 0.5 mL  and transfer to douncer. Repeat with another 0.5 mL to get all tissue.
SECTION: Tissue Preparation
9. Dounce: 5-7x loose, 5-7x tight.
SECTION: Tissue Preparation
10. Pour douncer content into falcon tube with sucrose/lysis buffer mixture and flip upside down a few times. Pour 1 mL of tube content back and forth from douncer to collect remaining nuclei.

SECTION: Tissue Preparation
11. Carefully layer the 8.5 mL  to the centrifuge tube, on top of sucrose.

SECTION: Tissue Preparation
12. Balance the tubes (≤5 mg difference).

SECTION: Tissue Preparation
13. Place tubes in ultracentrifuge, spin at 15500 rpm.

SECTION: Tissue Preparation
14. Carefully aspirate off supernatant without disturbing the pellet.

 


SECTION: Tissue Preparation
15. Soften pellet for 10 min in 50 µL.

SECTION: Tissue Preparation
16. Add 100 µL  (containing a nuclei  marker, such as Draq7) and filter using 70um pipette tip filters into coated tubes.

SECTION: Tissue Preparation
17. FACS sort, low flowrate, largest nozzle, cooling system on. The sucrose will cause problems at FACS. Avoid this by adding dilution buffer (with your nuclei marker), 100 µL at the time (usually need 300 µL-400 µL for dilution to avoid problems – add that even before starting to sort).

SECTION: Tissue Preparation
18. For single nuclei sequence using 10x: Pre-coat the pipette tips needed to load the 10x machine according to step 1. 
SECTION: Tissue Preparation
19. For RNA extraction: Pellet nuclei at 1300 x g. Remove supernatant and snap freeze for later RNA extraction (or resuspend in 350 µL directly. Use RNeasy micro columns. For very small volumes, resuspend in RLT without pelleting).
TITLE: Research on an Evolutionary Game Model and Simulation of a Cluster Innovation Network Based on Fairness Preference-new manuscript
AUTHORS: Li Chuanyun, Xia Cao, Ming Chi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The cluster innovation network is an important part of regional economic development. In addition, the fairness preference of internal innovators in the processes of investment and benefit distribution are particularly important for curbing "free riding" and other speculative behaviors and for creating a good cooperation environment. Therefore, taking a cluster innovation network constructed by the weighted evolutionary BBV model as the research subject, this paper constructs an evolutionary game model of a cluster innovation network based on a spatial public goods game and the theory of fairness preferences, which involves the processes of investment and payoff allocation. Using simulation analysis, this paper studies the evolution of innovators’ cooperative behaviors and benefits in cluster innovation network under the conditions of a fairness preference and a return intensity. The results show that an increase in the weight coefficient, gain coefficient and degree of differentiation between the previous income and current investment can effectively promote improvements in the level of enterprise cooperation. Indeed, the greater the weight coefficient, the gain coefficient and the degree of differentiation are, the more substantial the improvement in the level of enterprise cooperation will be. Moreover, an improvement in the differentiation of the breadth and depth of enterprise cooperation has an inhibitory effect on enterprise cooperation. Furthermore, whereas increases in regulation and gain coefficients can effectively promote enterprise cooperation. However, the increase in the weight coefficient has a different effect on enterprise benefit in terms of the breadth and depth of cooperation.Finally, we hope to improve the overall cooperation level and cooperation income of the network by deeply understanding the fair preferences of innovators in the processes of investment and benefit distribution, which is helpful for promoting the evolution and development of cluster innovation networks.</div></div>

[STEPS]
TITLE: Home isolation in case of possible COVID-19
AUTHORS: Lenny Teytelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is our family protocol for isolation in case one of us (parents) has symptoms which could be consistent with COVID-19.</div><div class = "text-block">After a light scare a few days ago that required SARS-CoV-2 testing, we realized just how much we did wrong in immediate response, despite 7 months of living in the pandemic.</div><div class = "text-block">This is a protocol for our family of two adults and two kids in a small 3-bedroom/1-bathroom apartment; please use it to make your own plans, as appropriate for your family and living situation.</div></div>

[STEPS]
?. At the onset of symptoms, start wearing a mask immediately, whether at home or in the car.
?. Schedule a SARS-CoV-2 test
?. Person with symptoms isolates in the parents' bedroom with the door always closed.
Move the folding table, monitor, chair to the kid's bedroom if needed for the other parent's work.
?. Wear a mask for restroom trips. Sanitize surfaces when done.
Have others wait   before using the restroom.Use a potty for urination throughout the day to minimize number of trips to the restroom.
?. Use Zoom/FaceTime to see kids/spouse; do not allow other family members into the bedroom.
?. Remember that SARS-CoV-2 PCR tests have a high false negative rate.If test results come back negative, continue isolation until symptoms are gone.
TITLE: Freezing Tissue in OCT using Isopentane
AUTHORS: Liz Tuck

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><h1>Introduction</h1></div><div class = "text-block">This protocol describes how to freeze tissues in OCT using dry-ice cooled isopentane (for downstream cryo-sectioning).</div><div class = "text-block"><h1></h1></div><div class = "text-block"><h1>Related Documents </h1></div><div class = "text-block">Risk assessment: WTSI-3200_ Freezing Tissue using Isopentane</div></div>

[STEPS]
?. On a downflow or fume hood: Place metal container on dry ice and half fill with isopentane.(use enough iso-pentane to completely immerse the tissue pieces).Iso-pentane is extremely flammable and may be fatal if swallowed or enters airways. To be used in a fume-hood or downflow table wearing correct PPE.
?. Cool 1 pair of forceps in the dry ice. Keep the other pair at room temperature. If used, place labelled cryotube on dry ice.
?. Put lumps of dry ice into the iso-pentane and monitor with the -100⁰C  thermometer until it measures -70°C.
?. Label a cryomold and foil with the sample id
?. Trim the tissue into a piece small enough to fit into the cryomold, if required.
?. Half fill the cryomold with OCT and position the tissue in the cryomold.Note: for spatial transcriptomics use small cryomolds (maximum 10 x10 mm)Ensure that the tissue is oriented correctly to achieve the desired cryosection. E.g. Place tissue with region of interest to section, pushed flat at the bottom of the mold. Ensure there is a thin layer of OCT at the bottom of the mold so the tissue is fully covered. Dotted line in the image above shows how cross-sections would be cut from the tissue. Sections would be generated from the bottom of the block upwards.
?. Add more OCT if needed to ensure the tissue is fully submerged and covered in OCT.
?. Using forceps, submerge the cryomold in the container of isopentane, chilled to -70°C on dry ice.
?. When completely frozen, wrap sample in foil (still in cryomold), and keep on dry ice. Alternatively, remove sample from cryomold and place in labelled cryotube on dry ice.
?. Transfer samples on dry ice, to labelled box in -80°C freezer.
?. If freezing more samples monitor the isopentane temperature and adjust as necessary.
?. Wearing cold-resistant gloves, carefully remove the metal container of iso-pentane from the dry ice. The iso-pentane and dry ice can be left with the lid off, in a fume hood or downflow table to evaporate – ensure it is clearly labelled.Alternatively, the iso-pentane can allowed to warm to -10C, then decanted to a suitable bottle for re-use. The bottle must be labelled and kept in a fume hood/ downflow table, with the lid loosened until the iso-pentane returns to room temperature.
TITLE: Transforming yeast
AUTHORS: Brian    Teague

[DESCRIPTION]
In the same way that we transformed E. coli by making them take up a plasmid, we will transform yeast by inducing them to take up both a plasmid (to cut your genomic location) and some linear DNA to repair that location.

  


[STEPS]
1. Compute the volume of your Cas9 plasmid that contains 1 µg of DNA.
Compute the volume of your URA3 PCR product that contains 500 ng of DNA.
If you don't have enough DNA for one or both of these, check with an instructor.


2. Immediately upon retrieving a tube of yeast cells from the -80 °C freezer, thaw them rapidly by putting them in the 42 °C water bath for 30 s

3. Centrifuge 13.000 x g, 2 min. Remove and discard the supernatant.

4. Add the following to the tube of yeast cells in order:
260 µL  50 Mass / % volume 
36 µL  1 Molarity (M) 
50 µL  2 mg/mL
Enough plasmid DNA to equal 1 µg of DNA (computed above)
Enough PCR product to equal 500 ng of DNA (computed above)










5. Vortex vigorously to resuspend the pellet in the transformation mix. 

 

6. Incubate in the 42 °C water bath for 30 min

 

7. Centrifuge 13000 x g, 30 s . Aspirate the supernatant.

8. Pipette 1 mL of sterile water into the transformation tube. Stir the pellet with a micropipette tip to break up the cell pellet, then vortex to thoroughly resuspend the pellet.

9. Pour 5-10 glass beads on a Uracil dropout plate. Pipette 100 µL of cells onto it. Shake the plate to spread the cells out.

10. Pour 5-10 glass beads on a Leucine plate. Pipette 100 µL of cells onto it. Shake the plate to spread the cells out.

11. Incubate 48-72 hours upside down at 30 °C.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
Needed:<br /><ul><li>0.2 &micro;m filtered seawater with iron chloride</li><li>Mg-EDTA-Ascorbate (Oxalate) buffer</li><li>DNase I</li><li>EDTA</li><li>EGTA</li><li>Centrifugal concentrators</li><li>Resuspension buffer</li><li>Wizard prep column</li><li>Wizard prep resin</li><li>Quant-iT dsDNA Pico Green assay kit</li></ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Isolated human islets were dispersed into single cells using Accutase (Millipore), ~1500-2000 islet equivalents (IEQ)/ml. 20,000-65,000 islet cells were transferred to individual wells of a 96 well culture plate (CELLSTAR, Greiner Bio-one) for immediate staining for flow cytometry analysis or for culture prior to staining. Isolated human islet cells were cultured in the presence or absence of the HS mimetics heparin (a highly sulfated HS analogue from porcine intestinal mucosa), BT548 (a glycol split low molecular weight heparin (LMWH; 3 kDa) lacking anti-coagulant activity) or PI-88 (Progen Pharmaceuticals Limited,) at 50 μg/ml for 2 days in 5% CO<sub>2</sub>, 95% air at 37ºC. In some studies islet cells were acutely treated with 30% H<sub>2</sub>O<sub>2 </sub>(Chem-Supply) as a source of reactive oxygen species (ROS) for 5 min on day 0 or after culture for 2 days with/without HS mimetics. Beta cells were identified by staining with Newport Green (NG; 10 μmol/L; Invitrogen, Molecular Probes), a fluorescent probe that detects zinc in the insulin granules of beta cells. Damaged and dying islet cells were assessed using 7-Aminoactinomycin (7AAD, 10 μg/ml; Life Technologies) or by Sytox green (31.25 nmol/L; Invitrogen, Molecular Probes) uptake. Cells were analyzed using a BD LSRI flow cytometer and CellQuest™ Pro software (version 6.0; BD Biosciences).</p>

[BEFORE_START]
<p>Materials:</p>
<ol>
<li>Prepare:</li>
</ol>
<p> </p>
<p>(i)<u> PBS/3mM EDTA:</u></p>
<p>112 mg EDTA (AJAX #180) in 100 ml PBS, sterile filter using 0.2 μm disposable filter.</p>
<p> </p>
<p>(ii)<u> Beta cell culture medium:</u></p>
<p>RPMI 1640 (Sigma #R0883), 200 ml</p>
<p>Heat-inactivated fetal calf serum (HIFCS), 20 ml</p>
<p>L-Glutamine (Gibco # 25030081 200 mM) 2 ml (final 2 mM)</p>
<p>Penicillin G (MP Biomedicals #02194537), 0.06 mg/ml</p>
<p>Streptomycin (Sigma #S9137), 0.10 mg/ml</p>
<p>Neomycin (Sigma #N6386), 0.10 mg/ml</p>
<p> </p>
<ol start="2">
<li>Other reagents:</li>
</ol>
<p>Accutase, Millipore #SCR005</p>
<p>Cell culture plates: Cellstar #650180 (Greiner Bio-one)</p>
<p>Newport Green DCF diacetate (Newport Green), Invitrogen, Molecular Probes #N7991</p>
<p>7-Aminoactinomycin (7AAD) Life Technologies #A1310</p>
<p>SYTOX Green, Invitrogen, Molecular Probes #S7020</p>
<p>Hydrogen peroxide (30% w/w), Chem-Supply Pty Ltd (Australia) #HA154-500M</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em><strong>Making a Phylogenetic Tree</strong></em></p>
<p> </p>
<p>In this procedure we are going to build a phylogenetic tree! Throughout I'll refer to scripts available on my github here. We will be using the 16 ribosomal proteins used in Hug et al. 2016. The HMM models for these are available on my github in a file called `hug_ribosomalmarkers.hmm`. We will be running this tutorial using the genomes and files found in the folder `Example`.</p>
<p> </p>
<p>As an overview I will be explaining</p>
<p>1. How to generate gene predictions using prodigal for genomes of interest</p>
<p>2. How to identify ribosomal proteins using an hmm model</p>
<p>3. How to align, trim, and concatenate proteins for a phylogenetic tree</p>
<p>4. building a phylogenetic tree</p>
<p> </p>
<p>To cite this workflow reference this paper:</p>
<p>Graham, E. D., J. F. Heidelberg, and B. J. Tully. 'Potential for primary productivity in a globally-distributed bacterial phototroph.' <em>The ISME journal</em> (2018)</p>
<p>doi: <a href="https://doi.org/10.1038/s41396-018-0091-3" target="_blank" data-track="click" data-track-action="view doi" data-track-category="article body" data-track-label="link" rel="noopener noreferrer">https://doi.org/10.1038/s41396-018-0091-3</a></p>

[BEFORE_START]
<p>Before Starting Be sure you have all the required Pre-Requisites. </p>
<p> </p>
<p>Python2.7</p>
<p>BioPython</p>
<p>HMMER</p>
<p>Prodigal</p>
<p>BinSanity</p>
<p>Muscle</p>
<p>TrimAL</p>
<p>FastTree</p>
<p> </p>
<p>See <a href="https://github.com/edgraham/PhylogenomicsWorkflow/tree/master" target="_blank" rel="noopener noreferrer">this github page</a> for links to all dependencies.</p>
<p> </p>
<p>To cite this workflow reference this paper:</p>
<p>Graham, E. D., J. F. Heidelberg, and B. J. Tully. 'Potential for primary productivity in a globally-distributed bacterial phototroph.' <em>The ISME journal</em> (2018)</p>
<p>doi: <a href="https://doi.org/10.1038/s41396-018-0091-3" target="_blank" data-track="click" data-track-action="view doi" data-track-category="article body" data-track-label="link" rel="noopener noreferrer">https://doi.org/10.1038/s41396-018-0091-3</a></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.