eamag/protocols-with-steps huggingface dataset:
TITLE: Single cell dissociation of brain organoids
AUTHORS: michela.deleidi, María José Pérez J., Hariam Raji

[DESCRIPTION]
This protocol details about single cell dissociation of brain organoids.

[STEPS]
SECTION: Single cell dissociation of brain organoids
1. Mix 500 µL DNAse with 5 mL Papain. 
 



SECTION: Single cell dissociation of brain organoids
2. Transfer single or pooled organoid to 60 mm dish.
SECTION: Single cell dissociation of brain organoids
3. Aspirate excess media, add 2.5 mL Papain + DNAse solution.

SECTION: Single cell dissociation of brain organoids
4. With a razor blade mince organoid (<1 mm).
SECTION: Single cell dissociation of brain organoids
5. Transfer plate to an orbital shaker   (inside incubator).

SECTION: Single cell dissociation of brain organoids
6. With 1-mL pipette dissociate pieces (Mix up-down 30 times).
SECTION: Single cell dissociation of brain organoids
7. Put in orbital shaker 20 min.

SECTION: Single cell dissociation of brain organoids
8. In the meantime, add 5 mL Earle´s medium + 3 mL Inhibitor to a 15-mL conical tube.

SECTION: Single cell dissociation of brain organoids
9. Remove samples from the orbital shaker. With a 1-mL tip, mix up-down 30 times.
SECTION: Single cell dissociation of brain organoids
10. Take 2 mL (upper part) into new tube using a 40 µm cell strainer. Wait 1-3 min to debris to settle.

SECTION: Single cell dissociation of brain organoids
11. Transfer cell suspension to the inhibitor tube. Invert to mix 5 times.
SECTION: Single cell dissociation of brain organoids
12. Centrifuge 300 rpm, 7 min.

SECTION: Single cell dissociation of brain organoids
13. Aspirate supernatant, resuspend in 500 µL to 1 mL 0.5% BSA-PBS (Up-down 30 times).

SECTION: Single cell dissociation of brain organoids
14. Filter the resuspended cells (900 µL) with a 30 µm cell strainer.

SECTION: Single cell dissociation of brain organoids
15. Count the cells for the final suspension and dilute. Resuspend at 1000 cells/μl in 0.04% BSA-PBS.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>For questions, send an email to info.demonsprotocol@gmail.com</p>
<p>The complete protocol and instructions on measurement and analysis of eye movements with the DEMoNS protocol.</p>
<p><br /><span style="color: #333333; text-transform: none; text-indent: 0px; letter-spacing: normal; font-family: arial; font-size: 13px; font-style: normal; font-weight: 400; word-spacing: 0px; float: none; display: inline !important; white-space: normal; orphans: 2; widows: 2; background-color: #ffffff; font-variant-ligatures: normal; font-variant-caps: normal; -webkit-text-stroke-width: 0px; text-decoration-style: initial; text-decoration-color: initial;">Quantitative saccadic testing is a non-invasive method of evaluating the neural networks involved in the control of eye movements. The DeMONS protocol is a standardized and reproducible protocol for infrared oculography measurements of eye movements and analysis, which can be applied for various diseases in a multicenter setting. In the accompanying manuscript in PLoS ONE, descriptive and reproducibility values are listed of all parameters.</span></p>
<p> </p>
<p><span style="color: #333333; text-transform: none; text-indent: 0px; letter-spacing: normal; font-family: arial; font-size: 13px; font-style: normal; font-weight: 400; word-spacing: 0px; float: none; display: inline !important; white-space: normal; orphans: 2; widows: 2; background-color: #ffffff; font-variant-ligatures: normal; font-variant-caps: normal; -webkit-text-stroke-width: 0px; text-decoration-style: initial; text-decoration-color: initial;">Download the file Demons_all_files for the whole protocol including the matlabfiles, or download files seperately in the 'Steps' tab<br /></span></p>

[GUIDELINES]
<p>For instructions on analysis with the accompanying matlabfiles, read the 'Instructions analysis' file.<br />For questions, send an email to info.demonsprotocol@gmail.com</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><u>Goal:</u></p>
<p>This document aims to standardize the protocol for the labeling of bacteria using the Sigma Aldrich kits PKH26 (red) or PKH67 (green).</p>
<p><u> </u></p>
<p><u>General considerations:</u></p>
<p><u></u>- The handling of the material should be done within a Biological Safety Cabin (BSC), according to the bacterial species to be used. For example, for manipulation of <em>M. leprae</em>, BSC Class 2 (Cabins for manipulation of pathogenic biological agent, using out-of-laboratory airflow, which provides protection to the material, the manipulator, and the working environment).</p>
<p> - Sterile materials (microtubes, tips and solutions) should be used.</p>
<p> - The kit is stored in a refrigerator.</p>
<p> - The volume of initial bacterial culture to be used in the labeling protocol should correspond to twice the amount of bacteria that will be used in the experiment, because there is loss of bacteria in the processing.</p>
<p> </p>
<p><u>Experimental proceedings:</u></p>
<p>1. Centrifuge the solution with bacteria (previously quantified for use in the experiment *) at 18,000 x g for 5 minutes to sediment the bacteria;</p>
<p>2. Discard the supernatant, being careful not to lose the pellet;</p>
<p>3. Resuspend the pellet in 1X sterile PBS (same volume as contained in the initial solution);</p>
<p>4. Centrifuge the solution with bacteria at 18,000 x g for 5 minutes;</p>
<p>5. Discard the supernatant;</p>
<p>6. Prepare PKH solution: 1μl of the fluorophore PKH + 99μl of diluent reagent (PKH Kit);</p>
<p>7. Resuspend the bacterial pellet in the 100 μl of the PKH solution.</p>
<p>8. Incubate for 15 minutes at room temperature protecting from the light, vortexing briefly every 5 minutes, being careful not to raise the liquid to the cap and to leak.</p>
<p>9. Add twice the volume of fetal bovine serum (200 ul) - to stop the reaction - and incubate at room temperature for 1 minute;</p>
<p>10. Centrifuge the solution with bacteria at 18,000 x g for 5 minutes;</p>
<p>11. Discard the supernatant and resuspend in the culture medium for the experiment;</p>
<p>12. Quantify the labeled bacteria before use.</p>
<p> </p>
<p>References:</p>
<p><u> </u></p>
<p>Adapted from:</p>
<p><a href="https://www.sigmaaldrich.com/catalog/product/sigma/pkh67" target="_blank" rel="noopener noreferrer">https://www.sigmaaldrich.com/catalog/product/sigma/pkh67</a></p>
<p><a href="https://www.sigmaaldrich.com/catalog/product/sigma/pkh26" target="_blank" rel="noopener noreferrer">https://www.sigmaaldrich.com/catalog/product/sigma/pkh26</a></p>

[STEPS]
TITLE: Troubleshooting guide for DDNS
AUTHORS: Joyce Akello, Alex Shaw, Catherine Troman, Erika Bujaki, Manasi Majumdar, Aine OToole, c.ansley, Zoe Vance, rachel.colquhoun, Javier Martin, Nick Grassly, Andrew Rambaut

[DESCRIPTION]
This document provides general guidance for troubleshooting problems encountered in the DDNS method. It outlines the factors that might influence the outcome of the DDNS PCR assay run, the common problems encountered, possible causes of the problems, and provides recommendations on corrective action and preventative action. 
This document is primarily developed for DDNS performed on stool samples, but included are other factors to consider for other sample types.

[STEPS]
TITLE: Refractive Index Matching - Ethyl Cinnamate
AUTHORS: Holly Myers, Daphne Toglia

[DESCRIPTION]
This protocol details index matching for delipidated whole mouse brain samples in ethyl cinnamate solution to prepare for lightsheet imaging. Upon completion of this protocol, the delipidated mouse brain should be a light amber color and appear translucent when immersed in ethyl cinnamate. This index matching protocol may be paired with a modified iDISCO delipidation protocol: Whole Mouse Brain Delipidation - Dichloromethane. Ethyl cinnamate is an inexpensive, effective, and lower-toxicity index matching option that may be used in place of index matching reagents typically paired with this delipidation protocol (Hildebrand et al, 2019). 

[BEFORE_START]
This protocol is designed to index match a delipidated whole mouse brain specimen. For delipidation methodology, reference protocol Whole Mouse Brain Delipidation - Dichloromethane.

[STEPS]
SECTION: Index Matching with Ethyl Cinnamate
2. Using a serological pipet, measure 10mL of Ethyl Cinnamate into a 20mL glass scintillation vial. Place the delipidated mouse brain specimen into the vial of Ethyl Cinnamate using a spatula.
SECTION: Index Matching with Ethyl Cinnamate
3. Protect the vial containing the brain submerged in Ethyl Cinnamate from light by covering the vial with aluminum foil or placing the vial in a box. Leave for 4320 min at Room temperature.
SECTION: Index Matching with Ethyl Cinnamate
1. Ethyl Cinnamate is typically stored at 4 °C . Thaw bottle at Room temperature for 240 min ahead of use, or until the reagent is no longer frozen.
SECTION: Assessing for Successful Index Matching
4. Assess the brain specimen for successful index matching. It may be helpful to temporarily transfer the brain to a clear petri dish and completely immerse in Ethyl Cinnamate for this step, using a spatula to gently manipulate the brain in the dish if necessary.

Successful index matching:
When totally immersed in Ethyl Cinnamate, the brain should have an amber color and appear transparent, without visibly opaque interior structures. It should be possible to view numbers, letters, or fine lines on a grid through the brain without warping.

 

Unsuccessful index matching:
Parts of the brain around the cortex may appear transparent, while the deeper structures may have an opaque appearance. It will be difficult to view letters, numbers, or other images through the brain.


 
    


SECTION: Assessing for Successful Index Matching
4.1. If the brain appears successfully index matched, it is now ready for light sheet imaging. After imaging, the brain may be stored in Ethyl Cinnamate for several months at Room temperature .

If the specimen isn’t properly index matched, consider changing the Ethyl Cinnamate and leaving it for another 1440 min  at Room temperature  before imaging. The time required for clearing and dehydrating is dependent on the size of the specimen -- the larger the specimen, the longer it should be washed in each reagent in order to allow the reagent to fully penetrate the tissue.
TITLE:        ELISA for quantification of Vascular endothelial growth factor A (VEGFA)  in cell culture supernatant, human serum or plasma
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-vascular endothelial growth factor A (VEGFA) coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human VEGFA 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-vascular endothelial growth factor A 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 VEGFA antibody.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP conjugate.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of VEGFA 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 VEGFA standard dilutions and the human VEGFA sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Born in Bradford’s Better Start: an experimental birth cohort study to evaluate the impact of early life interventions
AUTHORS: Josie Dickerson, Philippa K. Bird, Kate E. Pickett, Dagmar Waiblinger, Eleonora Uphoff, Dan Mason, Maria Bryant, John Wright, Rosie RC McEachan

[DESCRIPTION]
Background: Early interventions are recognised as key to improving life chances for children and reducing
inequalities in health and well-being, however there is a paucity of high quality research into the effectiveness of
interventions to address childhood health and development outcomes. Planning and implementing standalone
RCTs for multiple, individual interventions would be slow, cumbersome and expensive. This paper describes the
protocol for an innovative experimental birth cohort: Born in Bradford’s Better Start (BiBBS) that will simultaneously
evaluate the impact of multiple early life interventions using efficient study designs. Better Start Bradford (BSB) has
been allocated £49 million from the Big Lottery Fund to implement 22 interventions to improve outcomes for
children aged 0–3 in three key areas: social and emotional development; communication and language
development; and nutrition and obesity. The interventions will be implemented in three deprived and ethnically
diverse inner city areas of Bradford.
Method: The BiBBS study aims to recruit 5000 babies, their mothers and their mothers’ partners over 5 years from
January 2016-December 2020. Demographic and socioeconomic information, physical and mental health, lifestyle
factors and biological samples will be collected during pregnancy. Parents and children will be linked to their
routine health and local authority (including education) data throughout the children’s lives. Their participation in
BSB interventions will also be tracked. BiBBS will test interventions using the Trials within Cohorts (TwiCs) approach
and other quasi-experimental designs where TwiCs are neither feasible nor ethical, to evaluate these early life
interventions. The effects of single interventions, and the cumulative effects of stacked (multiple) interventions on
health and social outcomes during the critical early years will be measured.
Discussion: The focus of the BiBBS cohort is on intervention impact rather than observation. As far as we are aware
BiBBS is the world’s first such experimental birth cohort study. While some risk factors for adverse health and social
outcomes are increasingly well described, the solutions to tackling them remain elusive. The novel design of BiBBS
can contribute much needed evidence to inform policy makers and practitioners about effective approaches to
improve health and well-being for future generations.


[STEPS]
1.
TITLE: Senescence Cocktail; Zhang Lab
AUTHORS: Sakthikumar Mathivanan

[DESCRIPTION]
Modeling age-related neurodegenerative disorders with human stem cells is difficult due to the embryonic nature of stem cell derived neurons. We developed a chemical cocktail to induce senescence of iPSC-derived neurons to address this challenge. We first screened small molecules that induce embryonic fibroblasts to exhibit features characteristic of aged fibroblasts. We then optimized a cocktail of small molecules that induced senescence in fibroblasts and cortical neurons without causing DNA damage. The utility of the “senescence cocktail” was validated in motor neurons derived from ALS patient iPSCs which exhibited protein aggregation and axonal degeneration substantially earlier than those without cocktail treatment. Our “senescence cocktail” will likely enhance the manifestation of disease-related phenotypes in neurons derived from iPSCs, enabling the generation of reliable drug discovery platforms.

[STEPS]
TITLE: Floral dip transformation of Arabidopsis
AUTHORS: Meike Burow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Konrad's version of the floral dip method - still insprired by</div><div class = "text-block">"Floral dip: a simplified method forAgrobacterium‐mediated transformation ofArabidopsis thaliana"</div><div class = "text-block">Steven J. Clough  Andrew F. Bent (1998)</div><div class = "text-block"><a href="https://doi.org/10.1046/j.1365-313x.1998.00343.x" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1046/j.1365-313x.1998.00343.x</span></a></div></div>

[STEPS]
?. Day 1 - Agrotransformation Electroporation (2400V, 220ohm, 25µF, 1mm cuvette)Recover 3 hours with 1ml YEP in 15 ml tube at 28°CPlate on YEP + antibiotics Incubate 2 days at 28°C
?. Day 3-4 - Prepare agrosStart 5ml agro culture in YEP + antibiotics. Grow o/n at 28°C, 220 rpm.Take 1-2 ml culture and plate on YEP plates + antibiotics (let soak into plate). Grow o/n at 28°C.
?. Day 5-6 - TransformationScrape cells from plates and resuspend in 50ml 10mM MgCl2 ◦ 6H2O (2.03 g/L) + 5% sucrose (50g/L) + 0.005% SilwetL-77 (50µl/L).Pour solution into small bag.Place pot upside-down in the agro-solution, massage gently so even un-open buds gets ‘cracked’ a little. Cut small hole in the corner of the bag, and pour the agro-solution into the next bag (for the next plant). Leave the plant o/n in the dark (decreases immune system). Remove bags and let plants dry before moving them to growth chamber.
TITLE: The pipeline of Hi-C assembly of the Scapharca broughtonii genome
AUTHORS: Chang-Ming Bai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol include the detailed methods of Hi-C assembly of the </span><span style = "font-style:italic;">Scapharca broughtonii </span><span>genome</span></div></div>

[STEPS]
?. Run BWA (v0.7.10-r789) to align the Hi-C reads to the initially assembled S. broughtonii genome, and found the Hi-C reads mapped to the assembled genome.
bwa index -a bwtsw fastabwa aln -M 3 -O 11 -E 4 -t 2 fq1bwa aln -M 3 -O 11 -E 4 -t 2 fq2
?. Filter the mapped Hi-C reads obtalined in the step 1 using HiC-Pro (v. 2.10.0).
mapped_2hic_fragments.py -v -S -s 100 -l 1000 -a -f -r -o
?. Extract valid interaction pair reads according the HiC-Pro results.
?. Break the initial assembly to 300 bp, and then run LACHESIS (v2e27abb) for assembling based on Hi-C data.
?. Run LACHESIS (v2e27abb) to assemble corrected contigs obtained in step 4 into chromosome and modified manually.
(1) CLUSTER_MIN_RE_SITES = 22；(2) CLUSTER_MAX_LINK_DENSITY=2；(3) CLUSTER_NONINFORMATIVE_RATIO = 2；(4) ORDER_MIN_N_RES_IN_TRUN=10；(5) ORDER_MIN_N_RES_IN_SHREDS=10.
TITLE: Thawing of cells
AUTHORS: Bettina Ergün

[DESCRIPTION]
This method describes the process of thawing permanent cell lines

[STEPS]
2. Allow cryotubes to thaw for 1 min at RT, then thaw for approx. 2 min  in a water bath while swirling until the last ice disappears

3. Disinfect the outside of the tube with alcohol
4. Pipette the content into a Falcon tube containing 10 ml of pre-warmed growth medium 
CAVE: In case of very sensitive cells, the cell suspension can be transferred from the cryotube directly into the culture bottle without centrifugation
6. 300 x g, 4 min 

5. Aspirate supernatant and resuspent pellet in growth medium (for T75: 4 ml )

7. at the resuspented cell without bubbles to a culture flask prefilled with growth medium (for T75: 8 ml) 

the size of the culture flask depends on the number of cells:
for 1-2E+6 cells => 1x T75
1.
TITLE: Algae Agar Preparation
AUTHORS: Dylan J Baker, Melissa B Duhaime, A.j Wing

[DESCRIPTION]
This protocol details the making of algae concentrate agar for the growth of bacteria from Douglas Lake.

Protocol for class project in Microbes in the Wild: Advanced Environmental Microbiology Lab (EEB 447) course at University of Michigan Biological Station and on campus. 

[STEPS]
1. During the peak of a cyanobacterial bloom, bring a 100μm mesh out into the water. 
Collect as many colonies as possible using the mesh and collect them into three sterile 50 ml falcon tubes. 

2. 
Sterilize a conventional blender with 10% bleach. Rinse with 1-2L MilliQ water
Pour the contents of the three 50 ml falcon tubes into the blender. Blend on high for 2 minutes.
Add the contents of the blender into a sterile 1L round glass media bottle.
Add 15 grams of agar to the media bottle.
Bring up the mixture to 1L with MilliQ water
Steam autoclave at 121˚C for 30 minutes on Liquid Cycle 2
Pour agar into sterile petri dishes and allow to set. Store plates at 4˚C.
TITLE: RNAlater Recipe
AUTHORS: Rex Malmstrom

[DESCRIPTION]
This is a storage solution that stabilizes and protectsRNA while inactivating RNase

[GUIDELINES]
Note: If you read the patent application, they refer to an ammonium sulfate concentration of 700g per L. What they really mean is add 700g to 1L (including EDTA and sodium citrate at conc. listed above), so you end up with more than 1L total volume. 

Note: The pH should be ~7 using this recipe. The patent states that pH can range from 4-8; the preferred commercial pH is 5.2; I use pH 7 for bacterioplankton samples, but pH can be lowered by adding H2SO4.

Note:  Since this is a storage solution for RNA, use high quality reagents ("molecular biology" grade) when preparing the buffer.

[STEPS]
SECTION: Preparation
1. Prepare 0.5 M EDTA  
 
SECTION: Preparation
2. Prepare 1 M Sodium citrate  
 
SECTION: RNAlater recipe
3. In a 2 or 3 L flask mix 1,400 mL MilliQ water with 60 mL 0.5M EDTA and 37.5 mL 1M sodium citrate with stirring
SECTION: RNAlater recipe
4. While continuing to stir the solution, add 1.05 kg (= 1,050 g) Ammonium sulfate in 100 g amounts.  
 
SECTION: RNAlater recipe
5. Stir ~30 min on hot plate set to low 
30 min
 
SECTION: RNAlater recipe
6. Allow solution to cool at room temperature for at least 30min, but overnight on bench is best 
30 min
SECTION: RNAlater recipe
7. Filter solution through 0.2 μm filter to remove particles
SECTION: RNAlater recipe
8. Store at room temp or at 4°C
TITLE: Real-time qPCR
AUTHORS: Shiyi Wang

[DESCRIPTION]
Real-time qPCR

[STEPS]
1. **Prepare cDNA Samples** - Plate cDNA samples on a 96-well qPCR plate.
2. **Prepare Reaction Mix** - Combine the following in each well: - 5 μL Fast SYBR Green Master Mix (4385616, Applied Biosystems) - 3 μL nuclease-free water - 0.5 μL forward primer - 0.5 μL reverse primer - 1 μL cDNA sample
3. **Ensure Technical Replicates** - Plate each sample two to four times to ensure technical replicates.
4. **Prepare Negative Control** - Use a control sample consisting of water with primers and Master Mix as a negative control.
5. **Perform qPCR** - Collect cycle threshold (Ct) values for each well.
6. **Normalize Data** - Normalize Ct values to GAPDH as a housekeeping gene.
7. **Primer Sequences** - Forward (F) primer for Atg7: 5′- GTTCGCCCCCTTTAATAGTGC -3′ - Reverse (R) primer for Atg7: 5′- TGAACTCCAACGTCAAGCGG -3′
TITLE: Sexing bovine preimplantation embryos by PCR
AUTHORS: Yao Xiao, Froylan Sosa, Peter J. Hansen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify">This protocol for using PCR to sex bovine embryos are based on Y-specific and autosomal targets described by Park et al, Theriogenology 2001; 55: 1843-1853 but with updates according to current bovine genomic database in NCBI as of 2019.Note that assembly of the bovine Y chromosome is still in progress.</div><style>
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[STEPS]