eamag/protocols-with-steps huggingface dataset:
TITLE: Regulation of mitophagy by the NSL complex underlies genetic risk for Parkinson’s disease: Drosophila Husbandry, Assays and Immunohistochemistry
AUTHORS: Natalie J. Welsh, Alexander J. Whitworth

[DESCRIPTION]
This protocol describes Drosophila stocks and husbandry, Locomotor and lifespan assays and, Immunohistochemistry and sample preparation in order to assess the role of KAT8/KANSL1 in neuronal survival in vivo.

[STEPS]
SECTION: Drosophila stocks and husbandry
1. Raise flies under standard conditions in a humidified, temperature-controlled incubator with a 12h:12h light:dark cycle at 25 °C, on food consisting of agar, cornmeal, molasses, propionic acid and yeast.
 

SECTION: Locomotor Assay
2. Perform the startle induced negative geotaxis (climbing) assay using a counter-current apparatus.
SECTION: Locomotor Assay
3. Place 20-23 males into the first chamber, and tap to the bottom. 
Give 10 s to climb a 10 cm distance.
Repeat this procedure five times (five chambers), and count the number of flies that remain in each chamber. 
SECTION: Locomotor Assay
4. Normalize the weighted performance of several group of flies for each genotype to the maximum possible score and expressed as Climbing index as described previously in the manuscript by Greene et al., 2003. 

 



SECTION: Lifespan Assay
5. For lifespan experiments, grow flies at low-density under identical conditions as described in Step 1   .

SECTION: Lifespan Assay
6. Collect progeny under very light anaesthesia and keep in tubes of approximately 20 males each, around 50-100 in total.
SECTION: Lifespan Assay
8. Calculate percent survival at the end of the experiment after correcting for any accidental loss. 
SECTION: Sample Preparation and Immunohistochemistry
9. Dissect drosophila brains from aged flies and immunostain as previously described in the manuscript by Whitworth et al., 2005. 
 

SECTION: Sample Preparation and Immunohistochemistry
10. Dissect adult brains in PBS and fix in 4 % (w/v) formaldehyde for30 min on ice.

SECTION: Sample Preparation and Immunohistochemistry
13. Incubate tissues with anti-tyrosine hydroxylase (Immunostar Inc. #22491), diluted in 0.3 % (v/v) Triton X-100 plus 4 % (v/v) goat serum in PBS for 4320 min at 4 °C, then rinse 3 times 20 min with 0.3 % (v/v) Triton X-100 in PBS, and incubate with the appropriate fluorescent secondary antibodies 20 min at 4 °C. 

SECTION: Sample Preparation and Immunohistochemistry
14. Wash tissues 2 times in PBS and mount on slides using Prolong Diamond Antifade mounting medium (Thermo Fisher Scientific). 
SECTION: Sample Preparation and Immunohistochemistry
15. Image brains with a Zeiss LMS 880 confocal. Count tyrosine hydroxylase-positive neurons under blinded conditions.
SECTION: Lifespan Assay
7. Transfer flies every 2-3 days to fresh medium and record the number of dead flies. 
SECTION: Sample Preparation and Immunohistochemistry
11. Permeabilize in 0.3 % (v/v) Triton X-100 for 3 times 20 min. 

SECTION: Sample Preparation and Immunohistochemistry
12. Block with 0.3 % (v/v) Triton X-100 plus 4 % (v/v) goat serum in PBS for 240 min at Room temperature.
TITLE: Confounding by Indication in Observational Studies Investigating Glucocorticoid-Associated Adverse Events in Rheumatoid Arthritis: A Protocol for a Systematic Review
AUTHORS: Andriko Palmowski, Judith Oldenkott, Anne Pankow, Anne Beenken, Eric L Matteson, Frank Buttgereit

[DESCRIPTION]
-

[STEPS]
SECTION: Abstract
1. Background Glucocorticoids (GCs) are regularly used drugs in rheumatoid arthritis (RA), but they can cause adverse events (AEs). Observational studies provide important real-world evidence but can be biased. Observational studies investigating the association between AEs and GCs in RA are
particularly susceptible to confounding by indication as patients with higher disease activity are more likely to suffer from AEs and more likely to receive GCs.

Objective To assess how often observational studies in RA which investigate the association between GCs and certain AEs adjust for disease activity (such as the disease activity score-28 joints) and/or systemic inflammation (such as serum C-reactive protein levels).

Methods A systemic literature review will be conducted and reported in compliance with the 2020 PRISMA statement. 



SECTION: PDF Protocol
2.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
10g/L Fe Stock Solution for&nbsp;Iron Chloride Precipitation of Viruses from Seawater Protocol.

[GUIDELINES]
<strong>Notes:</strong><br /><br />Solution is acidic and should be handled with care.<br /><br /> Discard solution if a cloudy precipitate forms. Do not dilute solution as iron hydroxide precipitate will form quickly. <br /><br /> For use on board ship, it is best to pre-weigh iron chloride into 50ml centrifuge tubes and add water as needed during the cruise: pre-weigh 0.966g FeCl<sub>3</sub>&middot;6H<sub>2</sub>O into several 50ml tubes. Add 20ml MQ-H<sub>2</sub>O when ready to precipitate viruses in seawater.

[STEPS]
?. 
?. 
?.
TITLE: Total Phenolic Content (TPC)
AUTHORS: Jing Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The TPC from samples was determined by a colorimetric methodbased on the procedure described byZhou et al. [10]2 ml Folin-Ciocalteu (FC) reagent was added to 2 ml of diluted extracts.After 3 min, 750 µL of sodium carbonate anhydrous solution (7.5%, w/v) was added, and the mixture was adjusted to 10 ml with distilled water. After 2 h, the absorbance was recorded at 765 nm. Calibration curves were constructed with gallic acid as a standard at concentrations ranging from 0–100 mg/ml. ml. </div></div>

[STEPS]
TITLE: Safety protocols for Aquatic Microbial Ecology Research Group, Department of Microbiology, The University of Tennessee.
AUTHORS: Steven Wilhelm, Gary LeCleir, Ashley Humphrey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a collection of safety protocols as used by the Aquatic Microbial Ecology Research Group. Science & Engineering Room 637 (O); 634 (Lab)</div></div>

[STEPS]
TITLE: bright field pheromone imaging
AUTHORS: Serena Ding

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For imaging aggregation behaviour of 40 young adult C. elegans on agar using the Phoenix multi-worm tracker system. Worms are synchronised by bleaching and refeeding for 72 hours, and then 40 young adult hermaphrodites are transferred by glass pipette onto a 35 mm regular NGM plate for imaging for 1 hour at 25 fps.</div></div>

[STEPS]
?. [Imaging plate preparation (Day -7)]
A separate batch of imaging plates is poured exactly seven days before each imaging day and stored at 4°C.
Imaging plates are 35 mm Petri dishes containing 3.5 mL low peptone (0.013% Difco Bacto) NGMagar (2% Bio/Agar, BioGene) to limit bacteria growth.
?. [Imaging plate preparation (Day -1)]
Imaging plates are dried at 37°C overnight with the agar side down.
?. [Seeding imaging plate (Day 0, AM)]
The center of an imaging plate is seeded with a single 20 μL spot of cold diluted OP50 (OD=0.75) one to three hoursbefore imaging.
The overnight plate drying step allowed the bacteria to quickly dry atop the media in order to achieve a more uniform lawn by minimizing the “coffee ring” effect that would thicken the circular edge of the bacteria lawn.
?. [Bleach synchronising worms (Day -7 to -4)]
Bleach synchronise gravid hermaphrodites. Leave on rotator at 20 °C until use.
?. [Re-feed worms (Day -3, PM)]
Re-feed starved L1 worms onto 3-4 plates using a glass pipette. Incubate at 20 °C.
Culture plates are 55 mm Petri dishes containing 15 mL low peptone 0.013% Difco Bacto) NGMagar (2% Bio/Agar, BioGene), and seeded with OP50.
?. [Imaging (Day 0)]
Forty animals are transferred by a glass pipette onto the imaging plate in a small drop of M9, away from the bacteria lawn.
?. [Imaging (Day 0)]
Imaging commences 20 s after the vibration finish, on a custom-built six-camera rig (the Phoenix) equipped with Dalsa Genie cameras (G2-GM10-T2041). One-hour recordings with red illumination (630 nm LED illumination, CCS Inc.) are taken at 25 Hz using Gecko software (v2.0.3.1), whilst the rig maintain the imaging plates at 20 °C throughout the recording durations.
?. [Image data processing]
Images are segmented in real time by the Gecko software.
?. [Imaging (Day 0)]
Wash animals off of culture plates with M9 and collect in a 15 mL Falcon tube, wash in M9 twice, and aspire as much supernatent as possible after the last wash.
?. [Imaging (Day 0)]
After M9 is absorbed into the media following worm transfer in liquid, imaging plates containing the animals are subjected to a gentle vibration at 600 rpm for 10 s on a Vortex Genie 2 shaker (Scientific Industries) to disburse animals and synchronize aggregation start across replicates.
?. [Image data processing]
Automated animal tracking is performed post-acquisition using Tierpsy Tracker software.
TITLE: PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)
AUTHORS: Yin-Tse  Huang

[DESCRIPTION]
PCR mixture and condition (2X SUPERGREEN PCR MASTER MIX)

[STEPS]
1. PCR mixture for generic primers
 

2. PCR mixture for barcoded primers

 

2.1. PCR condition for barcoded primers
 

1.1. PCR condition for generic primers
TITLE: Bioinformatics analysis based on the results of LC-MS/MS
AUTHORS: Lihui Li

[STEPS]
?. [Hierarchical Clustering]
Cluster3.0 and the Java Treeview software were used to study protein relative expression data performing hierarchicalclustering analysis. Euclidean distance algorithm for similarity measure and average linkage clustering algorithm for clusteringwere selected when performing hierarchical clustering. A heatmap was created as a visual aid in addition to the dendrogram.
?. [GO and KEGG Pathway Annotation]
The protein sequences of differentially expressed proteins were in batches retrieved fromUniProtKB database in FASTA format; then the NCBI BLAST+ software was used to find homologue sequences and transferred the functional annotation to the studied sequences (http://www.blast2go.com), which include three major categories: biological processes (BP), molecular function (MF), and cellular components (CC). In our study, we retrieved the top 20 blast hits (E-value＜1e-3) for each query sequence and loaded them into Blast2GO10 (Version 3.3.5) for Gene Ontology (GO) mapping and annotation. Regarding Kyoto Encyclopedia of Genes and Genomes (KEGG), the FASTA protein sequences of differentially altered proteins between two groups were blasted against the online KEGG database (http://www.genome.jp/kegg/) and then KEGG12was subsequently used for mapping pathways. The significantly overrepresented pathways and GO terms were extracted and classified.The enrichment analysis was performed to further explore the impact of differentially expressed protein in cell physiological process and discover the mechanism of internal relations between them. GO enrichment on three categories (biological process, molecular function, and cellular component) and KEGG pathway enrichment were analyzed by the Fisher’ exact test based on the entire quantified protein annotations as the background dataset. To adjust derived p-values, Benjamini-Hochberg correction was further applied for multiple testing. Only functional categories and pathways with p-values≤0.05 were considered as significant.
?. [Protein-Protein Interact Network（PPI）]
The protein–protein interaction information of the studied proteins was retrieved fromIntAct molecular interaction database by their genesymbols or STRING software. The results were downloaded in theXGMML format and imported into Cytoscape software to visualize and further analyze functionalprotein-protein interaction networks. Furthermore, to evaluate the importance of the protein in the PPI network, the degree of each protein wascalculated.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
These are protocols from: <br /><br />Paul, J. H., and M. Weinbauer. 2010. Detection of lysogeny in marine environments, p. 30–33. In S. W. Wilhelm, M. G. Weinbauer, and C. A. Suttle [eds.], Manual of Aquatic Viral Ecology. ASLO.<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_030.pdf" target="_blank">published manuscript</a> for additional information.<br /><br />

[STEPS]
?.
TITLE: Immunoblot
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Immunoblot of rodent brain regions

[STEPS]
1. Put the frozen bulk dissections from a specific brain region in a tube with lysis buffer (50mM Tris HCl, pH 7.0; 150 mM NaCl; 5 mM EDTA; 1% SDS; NP40-1%) supplemented with protease inhibitors (Roche). 
2. Homogenize the tissue with syringes, pestles or a tissue homogenizer with drill. Keep on
ice during homogenization.
3. Clarify the sample by centrifugation at 13000 rpm for 30 min at 4ºC. Collect the
supernatant into a new tube.
4. Determine the protein concentration using the BCA method.
5. Resolve proteins in a 10 or 15 % polyacrylamide gel and transfer onto 0.45 µm nitrocellulose membranes (Amersham).
6. Block the membrane with 5% milk powder in PBS1X.
7. Incubate the membrane with the primary antibodies diluted as needed overnight at 4ºC. 
8. Incubate the membrane with the secondary antibodies goat anti-rat, donkey anti-rabbit and sheep anti-mouse (all 1:1000, from Amersham) 1h at RT. 
9. Image protein bands using either West Pico SuperSignal Substrate or SuperSignal West Femto (Thermo Fisher Scientific, 34080 and 34095, respectively) on an ImageQuant RT ECL imaging system (GE Healthcare).
10. Determine band densitometry by using ImageJ image analysis software (RRID:SCR_003070, https://imagej.net/). Normalize the levels of each protein of interest to β-actin expression.
TITLE: 3D Printed Face Shield with Closed Top
AUTHORS: Julia Roßmanith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for building face shields with closed tops using the print template created by Hanoch Hemmerich on Thingiverse.</div></div>

[STEPS]
?. Print the head part of the face shield with the 3D printer.
?. Place the overhead transparency or laminated laminating foil on the head part.
?. Put a strap through both ends of the head part of the shield to fix it on your head.
TITLE: Media Recipes
AUTHORS: Brian Smith, baltrus@email.arizona.edu micro.tucson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here you will find common media recipes used in the Baltrus Lab at the University of Arizona for growing mostly </span><span style = "font-style:italic;">Pseudomonas spp.</span><span> and </span><span style = "font-style:italic;">E. coli</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
TITLE: Ty + AS + Thymidine Protocol
AUTHORS: Eleanor Harman

[DESCRIPTION]
.

[STEPS]
SECTION: Media
2. Add 80% final volume of DI water to beaker
SECTION: Media
1. Yeast Extract 1.5g/500mL
Tryptone 2.5g/500mL
Thymidine 150mg/500mL
SECTION: Media
3. Add Yeast Extract, Tryptone, and Thymidine until fully dissolved
SECTION: Media
4. Adjust pH to 5.5
SECTION: Media
5. Autoclave on Liquid 20 minute cycle
TITLE: Denaturing agarose gels for large RNAs with glyoxal
AUTHORS: Stephen Floor

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">PAGE gels work great for separating small RNAs under 500 nt or so, but cannot separate larger RNAs. This protocol uses glyoxal to denature RNAs, which can then be run on an agarose gel and will migrate according to their length rather than secondary structure etc. These gels work great and are an easier alternative to making formaldehyde gels when trying to separate large RNAs. Glyoxal reversibly reacts with guanine, adenine and cytidine bases in RNA preventing secondary structure formation. It is critical to maintain acidic pH (< 7.0), as the glyoxylation reaction is reversible in basic conditions. </div><div class = "text-block">The glyoxal reagent is NorthernMax loading dye from Ambion. http://www.lifetechnologies.com/order/catalog/product/AM8551  </div><div class = "text-block"><span>There are two steps: pouring the agarose gel and glyoxylating the RNA to be loaded onto the gel. Note that the buffer for this is not TAE!  Further note that the loading dye already has EtBR in it so staining is unnecessary, and </span><span style = "font-weight:bold;">do not add stain to the gel</span><span style = "font-weight:bold;">.</span></div><div class = "text-block">References: </div><div class = "text-block"><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://link.springer.com/protocol/10.1385%2F0-89603-127-6%3A1</span></a></div><div class = "text-block"><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">http://cshprotocols.cshlp.org/content/2006/1/pdb.rec328</span></a></div><div class = "text-block">RNA: A Laboratory Manual by Rio, Ares, Hannon and Nilsen (CSHP) </div></div>

[STEPS]
?. [Glyoxylate RNA]
Mix 500ng to 1ug of RNA sample 1:1 with glyoxal loading dye in ~10 ul
?. [Glyoxylate RNA]
Mix the required amount of ladder 1:1 with glyoxal loading dye
?. [Glyoxylate RNA]
Incubate at 50 degrees for 30 minutes
Note: proceed to pouring gel while glyoxylation reaction is occuring
?. [Glyoxylate RNA]
Snap cool reaction by placing it on ice immediately from 50 degrees to minimize formation of secondary structure
?. [Pouring gel]
Make 1 liter of 1X BPTE buffer (won't use this much but can save the rest).
?. [Pouring gel]
Make a 1% agarose solution in BPTE and dissolve by microwave
do not add stain to the gel! stain is in the glyoxal loading dye
?. [Pouring gel]
Pour gel and let cool to RT
?. [Running gel]
Load samples after 1-2 minutes on ice
?. [Running gel]
Run the gel at around 5V per cm between electrodes (typically ~80V). This usually takes 1-2 hours.
[CRITICAL] Excessive heating may reverse glyoxylation and/or degrade RNA
?. [Running gel]
Image gel using the EtBR channel
TITLE: U Mass - Lactate dehydrogenase
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">
This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Lactate dehydrogenase test on display and run the analysis.
?. Collect and analyze the data.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is from:</p>
<p> </p>
<p>Mofiz E. et al., <a href="http://dx.doi.org/10.1186/s13742-016-0129-2" target="_blank">Genomic resources and draft reference assemblies of the human and porcine scabies mites, Sarcoptes scabiei var. hominis and var. suis</a>. <em>GigaScience</em>. 2016. DOI: 10.1186/s13742-016-0129-2 <a href="http://dx.doi.org/10.1186/s13742-016-0129-2" target="_blank">http://dx.doi.org/10.1186/s13742-016-0129-2</a>.</p>

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

[DESCRIPTION]
<p>We have three program files that produced all of the results we discuss in the paper. All are iPython Jupyter Notebook files. The names are: wasps_replicator_11-single_caste.ipynb, wasps_replicator_11.ipynb, and dothegraphics.ipynb. The main code is in wasps_replicator_11.ipynb, with the first file simply modified for convenience for running the case in which every agent has the same caste. The results here were obtained in order to assure the conclusion that social hierarchy is influencing the results. The third file, dothegraphics.ipynb, accesses the data in the .csv files produced by the first two files. Because this is straightforward we include the here, but do not discuss it further. So, our protocol amounts to little more than: put the files in the right directory structure, open the notebook file, edit it to set the desired parameter values (defaults are given), and run it. We note that the iPython Notebook files have been given extensive annotation for the sake of documentation. The right directory structure is this:</p>
<p> </p>
<p>codeandoutputs/</p>
<p>      code/</p>
<p>      outputs/</p>
<p> </p>
<p>And the three code files mentioned above (the *.ipynb files) go into the code/ directory and are run from there.</p>
<p> </p>

[STEPS]
TITLE: Crystallization of SARS-CoV-2 Mpro
AUTHORS: blake.h.balcomb, Peter Marples, Lizbé Koekemoer, Daren Fearon, Charlie Tomlinson

[DESCRIPTION]
The COVID-19 pandemic has highlighted the need to identify novel therapeutic interventions and strategies for pandemic preparedness against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). This protocol outlines the crystallization protocol and buffer conditions used to obtain reproducible SARS-CoV-2 Mpro crystals suitable for XChem fragment screening.

[STEPS]
SECTION: Crystallization experiment
3. Protein and buffer requirements:
43.2 µL5 mg/mL  
1.92 mL 
14.4 µL   seeds, dilution 1:250

SECTION: Crystallization experiment
5. Dispense 20 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 nL5 mg/mL    to each lens using the SPT mosquito.
Dispense  150 nL to each lens using the SPT mosquito.
Dispense  50 nL to each lens using the SPT mosquito.

Drop ratio: 3:3:1 ratio (150 nl   : 150 nl reservoir solution: 50 nl seeds)
Final drop volume: 350 nl
SECTION: Crystallization experiment
4. Crystallisation screen composition:
0.1 Molarity (M)  MES pH 6.7 
11% PEG 4000
5% DMSO

Stock solutions used:
1 Molarity (M)  MES adjusted to pH 6.7 with HCl
50% w/v PEG 4000
99.9% DMSO

 

SECTION: Crystallization experiment
7. Crystal form after ~24 h.
 


SECTION: Crystallization experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallization experiment
2. Prepare seed stock:
 
1: 250 dilution    seeds
SECTION: Data collection at Synchrotron
8. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.78
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
TITLE: 09 Expression
AUTHORS: TJUSLS China

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Make the big batch of bacteria culture for protein expression.</div></div>

[STEPS]
?. Add 1L LB media and then add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours).
1 L
37 °C
?. Induce the culture to express protein by adding 0.3 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask.
?. Put the liter flasks in 16°C shaking incubator for 16h.
16 °C
?. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 RPM for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time. Freeze the cell paste at -80°C.
500 µl
4 °C