eamag/protocols-with-steps huggingface dataset:
TITLE: Generation of membrane tubules pulled from giant unilamellar vesicles (GUVs)
AUTHORS: Javier Espadas, Aurelien Roux

[DESCRIPTION]
This protocol explains the methodology to generate lipid nanotubes pulled from giant unilamellar vesicles for fluorescence microscopy experiments.

[STEPS]
SECTION: Protocol
1. Mix DOPC, DOPS and Atto 647N DOPE at 59.9:40:0.1 mol% respectively in a final volume of 200 µL with chloroform and 0.5 g/L lipid final concentration in a glass vial.
SECTION: Protocol
2. Dry the lipid mixture in the glass vials for 120 min in a vacuum chamber forming the dried lipid films on the bottom of the glass vials.
SECTION: Protocol
3. Add 200 µL of the lipid films hydration buffer A to the glass vial containing the dried lipid films.

SECTION: Protocol
4. Vortex the glass vials until visually seeing complete resuspension of the dried lipid films in the solution (seen by an increase in the turbidity of the lipid solution) forming the multilamellar vesicles (MLVs).
SECTION: Protocol
5. Mix 10 µL of MLVs with 2 µL of silica beads in an Eppendorf tube.

SECTION: Protocol
6. Deposit 6 drops of 2 µL each containing the mixture of MLVs and silica beads on a parafilm slide placed in the bottom of a petri dish.
SECTION: Protocol
7. Dry the drops for 60 min in the vacuum chamber until the liquid is completely dried.

SECTION: Protocol
8. Take one dried drop from the parafilm and insert it into a small plastic tip cutted at the thin end containing 6 µL of 1 Molarity (M) trehalose solution until visually seeing how the dried beads get to the thin bottom.
SECTION: Protocol
9. Incubate the cutted plastic tip containing the drop and the trehalose for 15 min at 60 °C attaching it to the cap of an Eppendorf with 500 µL destilled water inside by doing a small hole in the cap and inserting the cutted plastic tip.

SECTION: Protocol
10. Passivate the microscopy chamber by adding 200 µL solution of 2 g/L BSA for 15 min.
SECTION: Protocol
11. Remove the cutted plastic tip from the Eppendorf and put the thin part of the cutted tip in contact with the microscopy chamber containing 200 µL of working buffer until visually seeing how the beads are transferred from the tip to the observation chamber.
 

SECTION: Protocol
12. Gently stir the microscopy chamber to promote the detachment of the hydrated lipid films from the silica beads, leading to the formation of the GUVs.
SECTION: Protocol
13. Place a closed micropipette in the micro-positioning system (MP-285, Sutter Instrument, Novato, CA, USA), and use it to approach the pipette to the GUV membrane.
SECTION: Protocol
14. Touch the GUV membrane with the pipette and then move back the pipette until a lipid nanotube is pulled from the GUV.
SECTION: Protocol
15. Wait until protein coverage reaches the steady state in both the GUV and the pulled membrane nanotube.
TITLE: RNA isolation for tissue
AUTHORS: Chin Yee Tan

[STEPS]
?. Homogenization in TriZolAdd 1mlper and homogenize using handheld homogenizer.
[Trizol]
[tissue]
?. Incubate at RT for to allow nucleoprotein complexes to dissociate.
?. Spin down at max speed in a chilled centrifuge for .
?. Carefully remove the top aqueous phase and transfer to a new Eppendorf tube. The interphase and bottom organic phase can be saved for DNA and protein respectively.
?. To the aqueous phase, add of 100% isopropanol, mix by inversion and incubate at for a minimum for .
500 µl
?. Spin down at max speed for to precipitate RNA.
?. Remove supernatant, and add to wash the pellet.
[75% Ethanol]
?. Add carefully, and vortex to mix well.
[Chloroform]
?. Spin down at max speed for  and remove supernatant.
?. Resuspend pellet in appropriate volume of nuclease free water.
TITLE: Bjerrum Schafer-Nielsen buffer, modified by DING LAB, v1.0
AUTHORS: Pingtao  Ding

[DESCRIPTION]
This is a modified transfer buffer recipe for a semi-dry Western blot.

[STEPS]
SECTION: proportion guide
1. 48 millimolar (mM)  
39 millimolar (mM)  
15 % volume  
pH 9.2

This recipe is modified from the original Towbin (1979) buffer, with increased Tris base but reduced glycine.
This recipe is suitable for semi-dry transfer, and ideal for SDS-PAGE and 2-D PAGE.

References:

Garfin DE and Bers G (1989). Basic aspects of protein blotting. In Protein Blotting: Methodology, Research and Diagnostic Applications, B.A. Baldo et al., eds. (Basel, Switzerland: Karger), pp. 5–41.

Towbin H et al. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76, 4350–4354.
SECTION: proportion guide
2. for 1 L 
add in a 2 L 
58.2 g 
29.3 g 
add up to800 mL , stir and mix well until all salts are fully dissolved
measure pH, and it should reach around pH 9.2 
top up with ddH2O to 1 L 
SECTION: proportion guide
3. before using, dilute the 10x stock buffer into 1x working buffer
for 1 L 
take 100 mL 
add 150 mL 
top up to 1 L 
 


SECTION: proportion guide
4. Tips (modified from Bio-Rad: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6211.pdf):
1. Use high-quality, analytical grade reagents to enable better buffer conductivity and hence better transfer.
2. If reusing the buffer, measure the pH before use and make sure it maintains at pH 9.2.
3. Do not further dilute the transfer buffer from 1x to the levels below the recommended concentration, because this decreases the buffering capacity of the buffer.
4. Do not adjust the pH of the buffer, because it can result in increased buffer conductivity, manifested by higher initial current output and decreased resistance.
5. There is no SDS in this recipe, but it can be added when it is necessary. Increasing SDS in the transfer buffer increases protein transfer from the gel but decreases the binding of the protein to the nitrocellulose membrane. In this case, nitrocellulose membrane can be substituted by PVDF membrane when SDS is used in the transfer buffer.
6. Addition of SDS increases the relative current, power, and heating during transfer, and may also affect the antigenicity of some proteins.
7. Increasing alcohol in the transfer buffer decreases protein transfer from the gel and increases the binding of the protein to the nitrocellulose membrane.
TITLE: UC Davis - Endoplasmic reticulum stress
AUTHORS: Fawaz G. Haj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:  </span></div><div class = "text-block">This test is designated to determine if rodents exhibit signs of endoplasmic reticulum stress, through evaluation of the activation state of the 3 sub-arms: PERK/EiF2α, Ire1α/sXBP1, and ATF6α pathways such. We will examine induction of ER stress in adipose and liver tissues.</div></div>

[STEPS]
?. Unless otherwise requested by the PI or stated in the protocol, mice will be euthanized using cervical dislocation.
?. Collect maximum blood from portal vein and isolate plasma according to standard protocols or as desired by the P.I.
?. Quickly collect tissues designated by the P.I. Each tissue should be divided into three portions, one portion should be snap frozen in liquid nitrogen, one portion should be kept into RNA later solution and the third one should be fixed into the appropriate fixative solution. Please note that the whole procedure of tissue collection should be done within 3 minutes maximum.
?. For western blotting, tissues will be lysed into the appropriate lysis buffer.
?. Total protein expression of XBP1, BiP, ATF6α, phosphorylation of PERK, Ire1α and EIF2α in adipose tissue and/or liver (or any other tissue if requested by the P.I.) will be determined according to the standard Western blotting protocols.
?. Note: Evaluation of the activation state of other component of the ER stress and ER stress-associated signaling, particularly the unfolded protein response (such as ERAD proteins, calnexin, etc...), JNK pathway or ER stress-induced apoptosis is also possible upon special request. Extra charges may apply. Gene expression of proteins involved in unfolded protein response and ER stress is also feasible if requested by the P.I. Extra charges may apply. Immunohistochemistry of ER stress markers could be performed on fixed tissues if desired by the P.I. Extra charges may apply.
TITLE: Human neuroblastoma cell line SH-SY5Y culturing
AUTHORS: Qing Wang

[DESCRIPTION]
This is the brief protocol for seeding and culturing human neuroblastoma SH-SY5Y cells.

[STEPS]
1. 1. SH-SY5Y cells (ATCC, Manassas, VA, USA, Cat# CRL-2266, RRID:CVCL_0019) were seeded at 70-80% density.
2. 2. Change medium to DMEM/F12 supplemented with 15% FBS, 1% penicillin-streptomycin and 10μM retinoic acid (11320-033, Gibco) for differentiation for four days
TITLE: Macroalgae Analysis
AUTHORS: Nohea Rodriguez, Kai Bloom, Mistie Tran

[DESCRIPTION]
Determine the abundance and biomass of the invasive macroalgae species, Turbinaria and Sargassum. Then, identify marine species located on random samples of each species. Turbinaria decreases fish abundance and biodiversity, competing with native algae and coral for space and resources. Determine the effect on biodiversity if Turbinaria were to be removed from an ecosystem entirely. Under the guidance of Maya Lin, the ISP class will conduct transect data collection in Baie Vaiori, located in Mo'orea. The objective is to gather data on the spatial occupancy of algae. Additionally, algae will be gathered to bring back to the lab where they will be examined for any residing organisms.

[BEFORE_START]
Make sure participants are well equipped with proper snorkel gear. Before getting in water, have data table written out on slate clipboard for convience.

[STEPS]
SECTION: Algae Transect Survey
1. Split into five groups of four people.
SECTION: Algae Transect Survey
3. Swim to the group's transect location, approximately 5 meters apart.
SECTION: Algae Transect Survey
4. Begin 30 meter transect along reef crest, parallel to the shore.
5. Swim along transect tape and record the presence of rock, coral, Turbinaria, or Sargassum for every 10 centimeters of the transect. Take photos along the transect for later reference. (300 observations per 30m)
SECTION: Sample Inspection
8. Gather samples of Turbinaria and Sargassum into ziploc bags filled with seawater. Bring sample bags into dirt lab
SECTION: Sample Inspection
9. Dump out contents into a plastic tub, and strain water from algae into a colander.
6. Move 5 meters towards the shore, and repeat Step 4 and 5.
7. Repeat Step 6 (until you have three 30 m transects).
SECTION: Sample Inspection
10. Carefully inspect macroalgae bunches, searching for organisms.  
 

SECTION: Algae Transect Survey
4.1. Tie down transect tape to coral or rock and map out the transect trajectory and distance. Tie to the 1 meter mark so that your first observation is at 1 meters.


 


5.1.  
 
 
 

5.2. Record the species of coral if possible
SECTION: Algae Transect Survey
3.1. Groups should have received the location of their transect areas on shore
SECTION: Algae Transect Survey
2. Gather materials - Transect tape, slate clipboard, snorkel gear
SECTION: Sample Inspection
11. Photograph any organisms next to a ruler, against your white background

 

SECTION: Sample Inspection
12. Record the weight of each algae bunch, number, size, and type of critters present, stem length of each bunch
SECTION: Sample Inspection
13. Record field and lab data in separate spreadsheets
TITLE: 15-minute DNA extraction from beetle legs for PCR barcoding 
AUTHORS: Chase Mayers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This method allows for quick extraction of DNA from beetle specimens for routine PCR amplification of barcoding genes such as COI (cytochrome oxidase subunit 1) for beetle ID. It is likely to also be effective for other high-copy genes such as other mitochondrial genes or ribosomal genes. It may not yield enough DNA to amplify large fragments or low-copy genes, and is certainly not appropriate for next-generation sequencing. However, for quick ID of beetle vouchers (for example, for beetles from which fungal cultures were isolated in a mycological study) it's hard to beat a ~15 minute DNA extraction.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
?. Use a pipette to transfer  of PrepMan Ultra Sample Preparation Reagent into a small microcentrifuge tube. Label the tube appropriately.If possible, this should be done in a sterile environment and away from PCR equiptment to avoid contaminating the PrepMan reagent with unwanted DNA.
50 µl
?. Using sterile forceps, remove one leg (or multiple) from the beetle and transfer to the PrepMan reagant. Fresh, dried, and ethanol-pickled beetle specimens have all yielded usable results with varying success rates.
?. Using a sterile transfer needle (or other tool), thoroughly crush the leg(s) against the inner wall of the microcentrifuge tube.
?. Incubate the microcentrifuge tube at  for  .
100 °C
?. Optional: Centrifuge the microcentrifuge tube briefly and pipette the supernatant to a new microcentrifuge tube. Label appropriately.
?. The liquid in the tube is now ready to be used as template for PCR. It can be stored at  .Typically we used
-20 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The third report of the National Cholesterol Education Program guidelines recommend calculating the 10-year morbidity of atherosclerotic cardiovascular disease using risk calculators when treating high blood cholesterol in adults. We analyzed the changes in cardiovascular risk (CVR) among patients with human immunodeficiency virus (HIV) from México.</p>
<p><span style="text-decoration: line-through;"> </span></p>
<p>Materials and Methods</p>
<p>This observational, prospective cohort study compared the cardiovascular risk after one year of usual treatment among 460 HIV patients in México. Changes using the atherosclerotic cardiovascular disease risk estimator and changes in clinical outcomes were analyzed. The results were categorized as low or high CVR using a cut-off of 7.5%.</p>
<p>The cardiovascular risk at baseline showed a mean of 4.45% ± 6.1% (0.2% to 50.1%), which changed to 4.48% ± 5.4% (0.2% to 33.2one year later . After cardiovascular risk stratification at baseline, we found that 84.3% of patients had a low CVR, and 18% of this group had metabolic syndrome. Moreover, 15.7% of this population was at high cardiovascular risk and 47% had metabolic syndrome. Approximately 4.3% of patients had a CVR increase and 2.6% had a cardiovascular risk decrease. Of the total patients, 22.3% had metabolic syndrome at baseline.</p>
<p> </p>

[GUIDELINES]
<h1>Third Report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III) Final Report</h1>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Selection of a “housekeeping” miRNA to normalize qRT-PCR data
AUTHORS: Barbara Rizzacasa, Elena Morini, Ruggiero Mango, Chiara Vancheri, Simone Budassi, Gianluca Massaro, Sara Maletta, Massimiliano Macrini, Silvio D’Annibale, Francesco Romeo, Giuseppe Novelli, Francesca Amati

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The qRT-PCR expression analyses were performed on all the recruited patients (61 CAD, 38 AMI_T0 and 11 AMI_T1 patients respectively). Since the absence of a universally recognized miRNA with housekeeping properties, we have analyzed the amplification curve of 84 circulating microRNAs using the Human Serum & Plasma miScript miRNA PCR Array (MIHS-106ZA, QIAGEN).</div></div>

[STEPS]
?. Identifcation of a "housekeeping" microRNA among the 84 circulating of the Human Serum & Plasma miScript miRNA PCR Array (MIHS-106ZA, QIAGEN):
?. [Selection criteria:]
•	a Ct range
?. [Selection criteria:]
•	a 0.5FC1.5 (GeneGlobe Data Analysis Center)
?. [Selection criteria:]
•	evaluation of expression level by qRT-PCR in all the enrolled samples by using the comparative Ct method quantification (2-ΔCt method).
?. [Result:]
Based on the above criteria, miR-15b-5p did not show any significant differences in expression level among CAD, AMI_T0 and AMI_T1 patients and could be our internal control.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"><strong>Motivation: </strong>An important step of "metagenomics" analysis is the assembly of multiple genomes from mixed sequence reads of multiple species in a microbial community. Most conventional pipelines employ a single-genome assembler with carefully optimized parameters and post-process the resulting scaffolds to correct assembly errors. Limitations of the use of a single-genome assembler for </span><em style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">de novo</em><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;"> metagenome assembly are that highly conserved sequences shared between different species often causes chimera contigs, and sequences of highly abundant species are likely mis-identified as repeats in a single genome.<br /><br /><strong>Methods:</strong> </span><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">We modified and extended a single-genome and de Bruijn-graph based assembler, </span><a href="http://www.ebi.ac.uk/~zerbino/velvet/" target="_blank">Velvet</a><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">, for </span><em style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">de novo </em><span style="color: #333333; font-family: helvetica, sans-serif; font-size: 14px; line-height: 21px;">metagenome assembly. Our fundamental ideas are first decomposing de Bruijn graph constructed from mixed short reads into individual sub-graphs and second building scaffolds based on every decomposed de Bruijn sub-graph as isolate species genome.<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bnmc2w6.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bnkc2w6.png" data-ofn="fig1.png" /><br /></span>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><em>Amrasca biguttula biguttula </em>(Ishida) commonly known as cotton leafhopper is a serious pest of cotton and okra. Nutritional requirements or growth physiology for this insect has not been much known till date. We developed artificial membrane feeding method for leafhopper which can be further used for RNA interference studies. Main components of this diet were L-cysteine (5.0 mg), glycine (2.0 mg), nicotinic acid (1.0 mg), sucrose (500 mg), K<sub>2</sub>HPO<sub>4</sub>(50 mg), ZnCl<sub>2</sub> (0.04 mg), Thiamine HCl (0.25 mg), Vitamin B6 (0.25 mg), Becosules capsule powder (Pfizer Limited, USA) (2.0 mg), Green food dye (GanpatiSyn Food Colour, India) (2.0 mg) per 10ml of autoclaved double distilled water. To make diet accessible to the cotton leafhoppers the 1.5 ml of Microcentrifuge tube’s (MCT) cap (Tarsons Products Pvt. Ltd. India) was removed and diet was filled in 2 layers of parafilm sachet (diet was poured on first layer of parafilm on cap and covered with another layer of stretched parafilm) into cap. The MCT’s was cut 1/4<sup>th</sup> from bottom end, which was covered with muslin cloth (40 count mesh size). This MCTs with leafhopper was kept in incubator at 65-70% RH, 14:10 h light and dark photoperiod and 27± 2°C.<img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/qafh8k6.jpg" data-src="https://s3.amazonaws.com/pr-journal/qaeh8k6.jpg" data-ofn="Layout_Membrane_Feeding.jpg" /></p>

[BEFORE_START]
<ol>
<li>All apparatus (parafilm, knife, brush, forceps, scalpel, scissors, gloves) should be treated with DEPC solution prior autoclaving.</li>
<li>All apparatus (parafilm, knife, brush, forceps, scalpel, scissors, gloves) should be autoclaved before use.</li>
<li>Swab work bench with 75% ethanol</li>
</ol>

[GUIDELINES]
<ol>
<li>Carefully dispense the diet into the parafilm sachet so that there should not be any bubble formation.</li>
<li>Always use sterile knife to cut eppendorf tube.</li>
<li>Muslin cloth of optimum size is cut to cover the opening of MCT so that it does not form any fold.</li>
<li>Upright position of eppendorf tube with diet side facing upwards must be considered.</li>
<li>Diet should be sterilized using filter sterilization (0.22 µm MCE hydrophilic membrane HIMEDIA) method to remove any bacterial contamination.</li>
<li>The diet after filter sterilization, should be kept at 70°C for 15 minutes for dsRNAses deactivation,</li>
<li>Do not release more than six leafhoppers in one tube to avoid congestion.</li>
<li>The diet should be regularly checked for any bacterial/fungal contamination before use.</li>
</ol>

[STEPS]
TITLE: Liquid Growth Medium - Yeast
AUTHORS: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald

[DESCRIPTION]
This protocol describes the steps to prepare liquid culture medium for Saccharomyces cerevisiae. 

[BEFORE_START]
Have the following solutions premixed:

Glucose 20% 500 ml solution:
Concentration: 200 g/l
mix 100 g Glucose in 500 ml deionized water (ddH2O)

Optional:
SC-xx 10x 100ml solution:
Concentration: 19.2 g/l
mix 1.92 g into 100 ml ddH2O

YNB 20x 100ml solution:
Concentration: 134.4 g/l
mix 13.44 g into 100 ml ddH2O

[STEPS]
SECTION: Preparation of 500ml liquid yeast growth medium
1. Compound medium for autoclave
SECTION: Preparation of 500ml liquid yeast growth medium
1.1. Fill a 500 ml flask with 450 mLddH2O. 
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 500ml liquid yeast growth medium
1.2. Add 3.36 g Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids (YNB)
SECTION: Preparation of 500ml liquid yeast growth medium
1.3. Add 0.86 g SC-XX powder. 
 

SECTION: Preparation of 500ml liquid yeast growth medium
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 500ml liquid yeast growth medium
3. When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
SECTION: Preparation of 500ml liquid yeast growth medium
4. 
 

SECTION: Preparation of 500ml liquid yeast growth medium
1.1. Fill a 500 ml flask with 325 mLddH2O. 
Add a magnetic stirring bar and place the flask on a stirring hot plate.
SECTION: Preparation of 500ml liquid yeast growth medium
1.2. Add 25 mLYNB 20x solution (Yeast Nitrogen Base with Ammonium Sulfate without Amino Acids).
SECTION: Preparation of 500ml liquid yeast growth medium
1.3. Add 100 mL SC-XX 10x solution.
 

SECTION: Preparation of 500ml liquid yeast growth medium
2. Autoclave for 15 min at 121 °C.
 

SECTION: Preparation of 500ml liquid yeast growth medium
3. When the medium cooled down to around 80 °C add 50 mL sterile Glucose 20%.
SECTION: Preparation of 500ml liquid yeast growth medium
4.