eamag/protocols-with-steps huggingface dataset:
TITLE: Cell culture, transfection, and imaging
AUTHORS: Will Hancock-Cerutti, Pietro De Camilli

[DESCRIPTION]
This protocol describes general procedures for culturing HeLa cells, transient transfection, and imaging using an Andor Dragonfly spinning disk confocal system.

[STEPS]
SECTION: General preparation
1. Culture the HeLa-M cells at 37 °C in 5% CO2 and DMEM containing 10% FBS, 100 U/ml penicillin, 100 mg/mL streptomycin, and 2 Milimolar (mM) L-glutamine (all from Gibco).
 





SECTION: General preparation
2. For live-cell imaging experiments, seed the cells on glass-bottomed dishes (MatTek) at a concentration of 35,000 cells per dish and transfect after 1440 min using FuGene HD (Promega) in Opti-MEM (Gibco).

SECTION: General preparation
3. Image the cells 1440 min after transfection.

SECTION: General preparation
4. Just before imaging, remove the growth medium and replace with prewarmed live-cell imaging solution (Life Technologies).
SECTION: General preparation
5. For lysotracker experiments, incubate the cells in 50 Nanomolar (nM) LysoTracker Red DND-99 (ThermoFisher) in complete DMEM for 30 min, wash twice with media, then image in live-cell imaging solution.

SECTION: General preparation
6. Perform all live-cell imaging at 37 °C and 5% CO2.

SECTION: General preparation
7. Perform spinning-disk confocal microscopy using an Andor Dragonfly system equipped with a plan apochromat objective (63×, 1.4 NA, oil) and a Zyla scientific CMOS camera.
SECTION: General preparation
8. For any given experiment, use the same exposure time, laser power, and gain for image acquisition to allow for quantitative comparison.
SECTION: Imaging of cells stably expressing STING-GFP
9. Generate the cells stably expressing STING-GFP as described elsewhere.
SECTION: Imaging of cells stably expressing STING-GFP
10. Culture the stable STING-GFP HeLa-M cells at 37 °C in 5% CO2 and DMEM containing 10% FBS, 100 U/ml penicillin, 100 mg/mL streptomycin, and 2 Milimolar (mM) Lglutamine (all from Gibco).

SECTION: Imaging of cells stably expressing STING-GFP
11. For experiments using siRNA, transfect 60 pmols of the indicated siRNA using 6 µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per dish according to manufacturer protocol. Image the cells 4320 min after siRNA transfection.

SECTION: Imaging of cells stably expressing STING-GFP
12. For experiments using cGAMP, transfect  50 μg/L of cGAMP using 18 µL Lipofectamine RNAiMax (ThermoFisher) in Opti-MEM (Gibco) per dish according to manufacturer protocol. Image the cells 840 min after transfection.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>TAP medium is the most widely used for growth of the green alga <em>Chlamydomonas reinhardtii</em>. Note the presence of acetate effects the photosynthetic ability of the alga and minimal medium should be used instead if quantifying effects of mutations or inhibitors on photosynthesis.</p>
<p> </p>
<p>Source:</p>
<p>from Gorman, D.S., and R.P. Levine (1965) <em>Proc. Natl. Acad. Sci. USA</em> <strong>54</strong>, 1665-1669.</p>
<p>http://www.chlamycollection.org/TAP.html</p>
<p> </p>

[GUIDELINES]
<p>From Gorman, D.S., and R.P. Levine (1965) <em>Proc. Natl. Acad. Sci. USA</em> <strong>54</strong>, 1665-1669.</p>
<p>This is probably the most widely-used medium at present for experimental work.</p>
<p> </p>

[STEPS]
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TITLE: Protocol for Nuclei Isolation and Automated 10X Genomics Single Cell 5&#39; Gene Expression for Human Ovary Explants
AUTHORS: Nicolas Martin

[DESCRIPTION]
This is the 10X Genomics Automated Single Cell 5' Gene Expression protocol using nuclei suspension isolated from fresh frozen human ovary explants.  The Chromium Connect (PN-1000171) was used from Gem generation to final gene expression libraries. 

[GUIDELINES]
This protocol needs prior approval by the users' institutional review board (IRB) or equivalent ethics committee(s).

[STEPS]
SECTION: Nuclei Isolation Protocol for Human Ovary Explants
1. Chapter 1—Single Cell Gene Expression & Chromium Fixed RNA Profiling of the protocol CG000505 REV A was used to isolate nuclei from frozen human ovary explants with the following modifications: 1) a cordless motor pestle (VWR, Catalog number 47747-370) was used for Step f, Page 30 and 2) the samples were incubated for 15 min on ice for Step h, Page 30. 

https://www.10xgenomics.com/support/single-cell-gene-expression/documentation/steps/sample-prep/chromium-nuclei-isolation-kit-sample-prep-user-guide


SECTION: Chromium Next GEM Automated Single Cell 5&#x27; Reagent Kits v2
2. The Protocol CG000384 Rev D was used to generate gene expression libraries from nuclei suspension inputs. 

https://www.10xgenomics.com/support/instruments/chromium-connect/chromium-next-gem-automated-single-cell-5-reagent-kits-user-guide-v-2-chemistry
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Provides a short introduction of using the MEGAHIT, IDBA-UD, and SPAdes assemblers.</p>

[GUIDELINES]
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/d9vc2w6.png" data-src="https://s3.amazonaws.com/pr-journal/d9vc2w6.png" data-ofn="Screen Shot 2016-07-12 at 1.52.03 PM.png" /></p>

[STEPS]
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TITLE: Quantitative Flow Cytometry (qFCM) protocols for end-to-end optimization of cross-platform extracellular vesicle studies
AUTHORS: Sean M Cook, Vera A Tang, Joanne Lannigan, Jennifer C. Jones, Joshua A Welsh

[DESCRIPTION]
Flow cytometry (FCM) is a common extracellular particles (EPs), including viruses and extracellular vesicles (EVs), characterization method. Frameworks such as MIFlowCyt-EV exist to provide reporting guidelines for metadata, controls, and
data reporting. However, tools to optimize FCM for EP analysis in a systematic and quantitative way are lacking. Here, we demonstrate a cohesive set of methods and software tools that optimize FCM settings and facilitate cross-platform comparisons for EP studies. We introduce an automated small particle optimization (SPOT) pipeline to optimize FCM fluorescence and light scatter detector settings for EP analysis and leverage quantitative FCM (qFCM) as a tool to further enable FCM optimization of fluorophore panel selection, laser power, pulse statistics, and window extensions. Finally, we demonstrate the value of qFCM to facilitate standardized cross-platform comparisons, irrespective of instrument configuration, settings, and sensitivity in a cross-platform standardization study utilizing a commercially available EV reference material. 

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<h1> </h1>
<p>This has been adapted by Kylie James in Dr Sarah Teichmann’s laboratory. Kylie has used this protocol many times for retrieval of lymphocytes from human colon (mLN, sigmoid, caecum and transverse) for 10X chromium genomics. FACS analysis shows a resultant population of pure lymphocytes with high viability. Stress signatures are evident in the single cell sequencing data.</p>

[STEPS]
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TITLE: B-PER Lysis--CHEM 584
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The Thermo Scientific B-PER Bacterial Protein Extraction Reagent enables mild extraction of proteins from bacteria (</span><span style = "font-style:italic;">E. coli</span><span>) without the need for mechanical disruption. The reagent may be used for soluble protein extraction and inclusion body purification from bacterial cell lysates. The B-PER Reagent with Enzymes is supplied with lysozyme and DNase I to improve the extraction efficiency of large (> 70 kDa) molecular weight proteins and proteins expressed in inclusion bodies.</span></div></div>

[STEPS]
?. [B-PER Lysis to extract soluble protein]
Pellet bacterial cells by centrifugation at 5000 × g in a tared tube for .
?. [B-PER Lysis to extract soluble protein]
Optional: Add of lysozyme and  of DNase I per  of B-PER Reagent added.
2 µl
2 µl
1 mL
?. [B-PER Lysis to extract soluble protein]
Add  of B-PER Reagent per gram of wet cell pellet. Pipette the suspension up and down until it is homogeneous.
4 mL
?. [B-PER Lysis to extract soluble protein]
Incubate  at room temperature.
?. [B-PER Lysis to extract soluble protein]
Centrifuge lysate at  high speed (~15,000 × g) for  to separate soluble proteins from the insoluble proteins.
Note: If a large percentage of over-expressed protein remains in the pellet, the protein of interest might be expressed in inclusion bodies.
?. [B-PER Lysis to extract soluble protein]
Decant your spent LB and determine the mass of your cell pellet.
?. [B-PER Lysis to extract soluble protein]
Freeze your pellet in the -20°C freezer overnight or quickly freeze your pellet in liquid nitrogen. When you are ready to proceed, thaw your cells at room temperature.
TITLE: Spatially variable gene detection
AUTHORS: Peter Kilfeather

[DESCRIPTION]
Spatially variable gene detection from Kilfeather, Khoo et al., 2024

[STEPS]
SECTION: Protocol
1. Spatially variable genes were identified using SpatialDE2 with default settings. Coordinates and full expression matrices of each brain were supplied, divided by cell type. To combine results from separate samples, a meta-analysis was performed on the raw P values from each sample, using Fisher’s method. The Benjamini & Hochberg correction for multiple comparisons was subsequently used and genes were considered spatially variable with an adjusted P value of < 0.01.
TITLE: Dual Optical Mapping of Action Potentials and Calcium Transients in the Mouse Heart during Optogenetic Stimulation of the ICNS
AUTHORS: Pradeep Rajendran, Guy Salama, Ching Zhu, Peter Hanna

[STEPS]
?. The animal is euthanized by a method approved by IACUC (Institutional Animal Care and Use Committee), the chest opened, the heart and lungs removed, and the aorta quickly cannulated on a Langendorff apparatus to perfuse the coronary vessels.  The cannulation step should occur in ice-cold Tyrode's solution (112mM NaCl, 5mM KCl, 25mM NaHCO3, 1mM KH2PO4, 1.2mM MgSO4, 1.8mM CaCl2, 50mM glucose) and completed within 10 minutes of euthanasia.
?. With a Langendorff apparatus, hearts can be perfused at either constant coronary pressure or constant flow rate.  Hearts were immobilized in a custom-built chamber to reduce motion artifact.
?. Hearts were stained with bolus injections of voltage-sensitive RH237 (30uL of 1mg/mL in DMSO, Thermo Fisher Scientific, S1109) and Ca indicator Rhod-2 AM (30uL of 1mg/ml in DMSO, Biotium, 50024) into the coronary perfusate.
?. Light from a 100-W tungsten lamp was collimated, passed through 530 ± 30 nm interference filters, split by a 560 nm dichroic mirror, and focused on the dorsal epicardial surface of the heart for excitation. Emitted fluorescence was collected with tandem camera lenses (50 mm f/1.2 mm, Nikon and 50 mm f/0.95, Navitar) and split with a 600 nm dichroic mirror.
?. The longer wavelength moiety, containing the Vm signal, was filtered between 610-750 nm and the shorter wavelength moiety, containing the intracellular Ca2+ signal, was filtered between 570-595 nm. The emitted fluorescence signals were recorded using 2 CMOS cameras (SciMedia, MiCAM ULTIMA) with a sampling rate of 2 kHz and 100 x 100 pixels with a 5 x 5 mm field of view. Pixel resolution of the images was 150 x 150 µm2. All stimulations were performed on the dorsal heart at 10 Hz, 10 ms, and 221 mW for 10 s. Data were acquired in 40 s intervals with 15 s collected before and after the stimulation.
?. Excitation-contraction uncoupling agents such as blebbistatin were not used.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>ABSTRACT</strong></p>
<p><strong>BACKGROUND: </strong>Transfusion therapy is associated with increased morbidity, mortality and costs. Conventional coagulation tests are weak bleeding predictors, poorly reflecting coagulation in vivo. Thromboelastometry (ROTEM) provides early identification of coagulation disorders and can guide transfusion therapy by goals, reducing blood components transfusion. <strong>Objective:</strong> The aim of this study is to describe coagulation profile of critically ill patients using ROTEM and evaluate the association between CCT and thromboelastometry. <strong>METHODS</strong>: This is a retrospective, observational study conducted in medical-surgical intensive care unit (ICU). Adult patients (≥18 years) admitted to ICU between November 2012 and December 2014, in whom ROTEM analyses were performed for diagnosis of coagulopathy, were included in this study. The first ROTEM and CCT after ICU admission were recorded simultaneously. Additionally, we collected data on blood components transfusion and hemostatic agents immediately after laboratory tests results. <strong>RESULTS</strong>: The study included 531 patients. Most ROTEM tests showed normocoagulability profile [INTEM (54.8%), EXTEM (54.1%) and FIBTEM (53.3%)] with divergent results in relation to CCT: low platelet count (51.8% in INTEM and 55.9% in EXTEM); prolonged aPTT (69.9% in INTEM and 63.7% in EXTEM) and higher INR (23.8% in INTEM and 27.4% in EXTEM). Approximately 16,7% of patients with normocoagulability in ROTEM received platelet concentrates and 10% fresh frozen plasma . <strong>CONCLUSION</strong>: The predominant ROTEM profile observed in this sample of critically ill patients was normocoagulability in contrast to CCT, suggesting coagulopathy and leading to a possibly unnecessary allogenic blood component transfusion. ROTEM test may avoid inappropriate allogeneic blood products transfusion in these patients.</p>

[STEPS]
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TITLE: TMT labelling
AUTHORS: Louise Uoselis

[DESCRIPTION]
TMT labelling

[STEPS]
1. Reconstitute lyophilised peptides in 25 µL by vortexing each sample for ~10 s, and then leaving to sit at Room temperature for 15 min. Each sample was then sonicated for 5 min in a waterbath sonicator in an ice slurry. 

2. Determine the peptide concentration of each sample spectroscopically
3. Aliquot10 µg of peptides for each sample into a LoBind microfuge tube and add 100 millimolar (mM) triethylammonium bicarbonate to a final volume of 100 µL for each sample. 

4. For the pooled batch control, divide 100 µg by the total number of samples, and aliquot that amount of peptides from each sample into one tube. Make up the pooled control volume to 100 µL with 100 millimolar (mM) triethylammonium bicarbonate (can lyophilise the samples to reduce the volume if the total pooled volume exceeds 100 uL). 

5. Bring the TMT labels to Room temperature. 

6. Reconstitute each label in acetonitrile as per the manufacturer’s instructions. 
7. Add the required volume of the designated label to each sample, and vortex each sample for ~ 5 s to mix. 

8. Leave samples to incubate at Room temperaturefor60 min (static).

9. Add hydroxylamine to a final concentration of 0.26%/sample, vortex each sample for ~5 s, and incubate at Room temperaturefor15 minto quench the TMT labelling reaction.

10. Combine the samples into their designated batches, and lyophilise the pooled samples. Seal each tube with parafilm and store at  -80 °C  for downstream processing.
TITLE: Copy of Protocol for the production of crude alcoholic extracts from native plants
AUTHORS: Marissol Leite, Hadda Tercya Lima Silva, Zanderluce Gomes Luis, Aline Coutinho Cavalcante, Caio Maximino, Diogenes Silva

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the method to obtaining crude alcoholic extracts from plant organs, such leaves and inflorescences. The used plants were the popularly called “João Brandinho” </span><span style = "font-style:italic;">Piper Callosum</span><span> and “Jambu” </span><span style = "font-style:italic;">Spilanthes acmella</span><span>. The crude extracts were used in our laboratory to anesthetize the native silver tetra fish </span><span style = "font-style:italic;">Ctenobrycon sp</span><span>. and Zebrafish </span><span style = "font-style:italic;">Danio rerio</span><span>, by immersion method.</span></div></div>

[STEPS]
?. Separate the leaves and inflorescences from plants with the help of scissors.
?. Rinse the material under running water and then with distilled water to remove debris.
?. Dry the material with paper towels to remove the excess of water.
?. Grind the leaves and flowers with a knife mill or blender and / or just cut with the help of scissors, in order to allow a better extraction of secondary compounds.
?. Weight the vegetable mass into an analytical balance and dilute in 70% alcohol.
?. After quantifying the vegetable mass, dilute and mash the crushed and / or cut material into 70% alcohol in the desired ratio, such as 1:1 (1 gram of vegetal material to 1 milliliter of 70% alcohol) until the thoroughly solution mix.
?. After maceration and dilution in 70% alcohol filter the obtained solution with the aid of filter paper in a beaker.
?. Identify the alcoholic crude extract and store in an amber glass in the refrigerator
TITLE: Disruption of Synechocystis cells
AUTHORS: iGEM Dusseldorf

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for disrupting Synechocystis cells to get soluble and non-soluble protein in different fractions</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Carry out all steps on ice</li><li style = "counter-reset:ol0;list-style-type:disc;">Thylacoid-buffer (TP)</li><li style = "counter-reset:ol0;list-style-type:disc;">    50 mM HEPES/NaOH</li><li style = "counter-reset:ol0;list-style-type:disc;"><span>    5 mM MgCl</span><span style = "vertical-align:sub;">2</span></li><li style = "counter-reset:ol0;list-style-type:disc;">    50 mM EDTA. pH 8</li><li style = "counter-reset:ol0;list-style-type:disc;">    1 Tablet Protease inhibitor (Roche complete ULTRA tablets) per 10 ml TP</li></ul></div><div class = "text-block"><span style = "font-style:italic;">Cell disruption</span></div><div class = "text-block"><span>Add 200 µl TP to a pellet of 20 ml Cell culture of OD</span><span style = "vertical-align:sub;">750</span><span> ~0,5 and resuspend the pellet</span></div><div class = "text-block">Add ca. 200 µl glas beads (1:1 mix of 0,1mm beads and 0,25 mm beads)</div><div class = "text-block">Use program 2 of the precellys to disrupt the cells at room temperature, put on ice immediately afterwards</div><div class = "text-block">Centrifuge for 5 min at 4°C and 4000 rpm to seperate glas beads and cellular waste from protein</div><div class = "text-block">Take supernatant (Whole cell extract 1)</div><div class = "text-block">Wash glas beads with 200 µl TP and repeat Precellys disruption</div><div class = "text-block">Centrifuge for 5 min at 4°C and 4000 rpm to separate glas beads and cellular waste from protein</div><div class = "text-block">Take supernatant (Whole cell extract 2)</div><div class = "text-block">Mix Whole cell extract 1 and 2</div><div class = "text-block">Optional: Centrifuge for 45 min at 21000 rpm and 4°C to seperate soluble from non-soluble protein. Soluble protein will contain Phycocyanin, non-soluble protien will contain Chlorophyll.</div><div class = "text-block">Freeze the protein until use, keep on ice when using it.</div></div>

[STEPS]
TITLE: Calibration Protocol - OD600 Inter-equipment Conversion with LUDOX
AUTHORS: Paul Rutten, Richard Tennant, Jacob Beal, Traci Haddock-Angelli, Natalie Farny, Geoffrey Baldwin, Marko Storch, Ari Dwijayanti

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>With this protocol you will use LUDOX CL-X (a 45% colloidal silica suspension) as a single point reference to obtain a conversion factor to transform absorbance (OD</span><span style = "vertical-align:sub;">600</span><span>) data from your plate reader into a comparable OD</span><span style = "vertical-align:sub;">600</span><span> measurement as would be obtained in a spectrophotometer. This conversion is necessary because plate reader measurements of absorbance are volume dependent; the depth of the fluid in the well defines the path length of the light passing through the sample, which can vary slightly from well to well. In a standard spectrophotometer, the path length is fixed and is defined by the width of the cuvette, which is constant. Therefore this conversion calculation can transform OD</span><span style = "vertical-align:sub;">600</span><span> measurements from a plate reader (i.e. absorbance at 600 nm, the basic output of most instruments) into comparable OD</span><span style = "vertical-align:sub;">600</span><span> measurements. The LUDOX solution is only weakly scattering and so will give a low absorbance value.</span></div><style>
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[STEPS]
?. [Data collection: OD 600 Reference point - LUDOX Protocol]
Add 100 μl LUDOX CL-X into wells A1, B1, C1, D1
?. [Data collection: OD 600 Reference point - LUDOX Protocol]
Add 100 μl of ddH20 into wells A2, B2, C2, D2
?. [Data collection: OD 600 Reference point - LUDOX Protocol]
Measure absorbance at 600 nm of all samples in the measurement mode you plan to use forcell measurements
?. [Data collection: OD 600 Reference point - LUDOX Protocol]
Record the data in your notebook or in the table below:   ABC1LUDOX CL-XddH2O2Replicate 13Replicate 24Replicate 35Replicate 4
ABC1LUDOX CL-XddH2O2Replicate 13Replicate 24Replicate 35Replicate 4
?. [Data collection: OD 600 Reference point - LUDOX Protocol]
Once you've collected your results, open this spreadsheet to process them.
?. [Data processing and analysis]
Yellow cells will automatically populate with values calculated from your raw data.
Here is an example of a completed table:
?. [Data processing and analysis]
Input your raw values from Step 5 into the blue cells in the table.
?. [Congratulations!]
You have now completed this calibration protocol
TITLE: 10x Protocols: Visium v2 CytAssist FFPE Library Construction -- University of Minnesota TMCs (CG000495 Rev C)
AUTHORS: IOx Genomics, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Protocols from 10x Genomics for Visium Spatial Gene Expression v2 chemistry on FPPE samples with the CytAssist component.

Protocol ID# (CG) and Revision used: 
    10x protocol CG000495, Revision C -- Library Construction with CytAssist

Note: These protocols may not be the current version offered by 10x Genomics but were used to produce the specific datasets connected to them. Please review the 10x Genomics support website for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
1. 10x protocol CG000495, Revision C (Library construction with CytAssist)
  

2. Additional Protocols/Resources
https://www.10xgenomics.com/support/spatial-gene-expression-ffpe