eamag/protocols-with-steps huggingface dataset:
TITLE: Adapted protocol to extract total RNA using TRIzol® (Invitrogen, Thermo)
AUTHORS: Talita Gagliardi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Total RNA extraction using TRizol reagent.</div><div class = "text-block">Attention: for nasopharyngeal aspirates, we use regular TRizol reagent in this assay, but for blood samples it is better to use a special one as "TRizol LS".</div></div>

[STEPS]
?. Incubate the homogeneized sample for 5min at r.t.
?. Add 150ul of chloroform
?. Shake the tube vigorously by vortex for 15sec.
?. Incubate for 3min at r.t.
?. Centrifuge for 15min at 12,000 x g and 4ºC.
?. The mixture separates into red lower phenol-chloroform phase, and interphase, and a colorless upper aqueous phase. RNA remains in the upper phase while the DNA and proteins in the interphase.
?. Place the aqueous phase for a new microfuge tube.RNA IsolationRNA precipitation
?. Add 375ul of Isopropanol.
?. Homogeneize by vortex.
?. Incube for 1h at r.t.
?. Centrifuge for 30min at 12,000 x g and 4ºC.
?. Discard the supernatant.RNA wash
?. Add 750ul of Ethanol 75%.
?. Homegeneize by vortex.
?. Centrifuge for 5min at 7,500 x g and 4ºC.
?. Discard the supernatant.
?. Ressuspend the RNA pellet in 30uL of RNAse-free H2O at 55-60ºC.
?. Homogeneize for 5 min by vortex.
?. Store the RNA at -70ºC.
?. In a microfuge tube: 750ul of TRIzol  (Invitrogen, Thermo)+ 250ul of sample
TITLE: Transfection of primary endothelial cells with siRNA
AUTHORS: Thomas Mudersbach, Daniel Siuda, Karin Kohlstedt, Ingrid Fleming

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

[STEPS]
?. [Preparing the endothelial cells for transfection (12-well plate)]
Cells were seeded the day before transfection at a density of 3 x 104 cells/cm2.
?. [Transfection ]
Cells were washed twice with PBS and than starved for at least in serum-free basal MCDB131 Medium at before incubation is finshed prepare the transfection mixture (volumes are for a single well of a 12-well plate)in one tube (A) add of siRNA (50 pmol stock) to Opti-MEMin a second tube (B) add of Lipofectamin RNAiMax into Opti-MEMMix each tube gently and incubate at room temperatur for After incubation the siRNA solution from tube A was added to the lipofectamin solution in tube B and incubated for at room temperatureof the siRNA/lipofectamin solution was added to each wellAfter to the medium was changed to endothelial cell growth mediumThe endothelial cells were incubated for at the washed with PBS and snap frozen in liquid nitrogen for further analysis
500 µl
1 µl
125 µl
3 µl
125 µl
225 µl
TITLE: Purification of GST-LC3B
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes the purification of GST-LC3B.

[STEPS]
SECTION: Purification of GST-LC3B
1. To purify GST-LC3B, insert human LC3B cDNA in a pGEX-4T1 vector. Note, the last five amino acids of LC3B are deleted to mimic the cleavage by ATG4.
SECTION: Purification of GST-LC3B
2. After the transformation of the pGEX-4T1 vector encoding GST-LC3B in E.coli Rosetta (DE3) pLysS cells, grow the cells in LB medium at 37 °C until an OD600 of 0.8-1 and induce the protein expression with 1 millimolar (mM) IPTG for 240 min at 37 °C.
SECTION: Purification of GST-LC3B
3. Collect cells by centrifugation and resuspend in lysis buffer.

Lysis buffer: 
 
 
SECTION: Purification of GST-LC3B
4. Sonicate cell lysates.
SECTION: Purification of GST-LC3B
5. Clear lysates by centrifugation at 140000 x g, 30 min, 4 °C in a Beckman Ti45 rotor.
SECTION: Purification of GST-LC3B
6. Collect and incubate the supernatant with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind GST-LC3B.
SECTION: Purification of GST-LC3B
7. Centrifuge the samples to pellet the beads and remove the unbound lysate.
SECTION: Purification of GST-LC3B
8. Wash beads twice with wash buffer, once with high salt wash buffer and two more times with wash buffer.

Wash buffer 
 

High-salt wash buffer 
 
 
SECTION: Purification of GST-LC3B
9. Elute the proteins 120 min with 20 millimolar (mM) reduced L-glutathione in 50 millimolar (mM) HEPES pH 7.4, 300 millimolar (mM) NaCl, 1 millimolar (mM) DTT buffer.
SECTION: Purification of GST-LC3B
10. Collect the supernatant, filter through a 0.45 µm syringe filter, concentrate using a 10 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 75 16/600 column (Cytiva).
SECTION: Purification of GST-LC3B
11. Elute the proteins with SEC buffer.

SEC buffer 
 
 
SECTION: Purification of GST-LC3B
12. Analyze fractions by SDS-PAGE and Coomassie staining.
SECTION: Purification of GST-LC3B
13. Pool fractions containing purified GST-LC3B protein.
SECTION: Purification of GST-LC3B
14. After concentrating the purified protein, aliquot the protein and snap-frozen it in liquid nitrogen. Store proteins at -80 °C.
SECTION: Purification of GST-LC3B
4.1. Sonicate cell lysates twice for 30 s (1/2).
SECTION: Purification of GST-LC3B
4.2. Sonicate cell lysates twice for 30 s (2/2).
TITLE: Regional Mouse Brain Analysis (Modified QUINT)
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This is series of protocols that has been adapted from published and unpublished protocols broadly referred to as the QUINT workflow. Note that the original QUINT workflow was generated by Yates and colleagues, and all credit for development of these programs goes to that team. References for each software is listed below.

Yates, S. C. et al. QUINT: Workflow for Quantification and Spatial Analysis of Features in Histological Images From Rodent Brain. Frontiers in neuroinformatics 13, 75, doi:10.3389/fninf.2019.00075 (2019).

Puchades, M. A., Csucs, G., Ledergerber, D., Leergaard, T. B. & Bjaalie, J. G. Spatial registration of serial microscopic brain images to three-dimensional reference atlases with the QuickNII tool. PloS one 14, e0216796, doi:10.1371/journal.pone.0216796 (2019).

VisuAlign, RRID:SCR_017978

1.	QuPath: https://qupath.github.io/ 
2.	QuickNII: https://www.nitrc.org/projects/quicknii (SELECT ABA Mouse Edition)
3.	Visualign: https://www.nitrc.org/projects/visualign/
4.	Qmask: \\pn.vai.org\projects_secondary\henderson\NOAH\QMask-Standalone
5.	Nutil: https://www.nitrc.org/projects/nutil/

[GUIDELINES]
Purpose 

The purpose of this workflow is to enable mouse brain segmentation, registration and quantification of regional signal. The simplest segmentation is done in QuPath because this program handles whole slide images and has good segmentation algorithms. Registration is done using 3 programs: QuickNII (aligns to a 3D atlas, typically the 2017 CCFv3 Allen Brain Atlas), Visualign (allows for non-linear warp transformation of the atlas to match sections), QMask (masks each side of the brain to allow for bilateral assessment of brain regions). Segmentations and registrations are then brought together in Nutil, enabling the generation of quantitative measures for every region of the brain.

Necessary Programs and Locations

QuPath: https://qupath.github.io/ 
QuickNII: https://www.nitrc.org/projects/quicknii (SELECT ABA Mouse Edition) 
Visualign: https://www.nitrc.org/projects/visualign/
Qmask: \\pn.vai.org\projects_secondary\henderson\NOAH\QMask-Standalone 
Nutil: https://www.nitrc.org/projects/nutil/



Folder Organization

The programs for registration and quantification rely on having exact file paths from which to call the data. Therefore, it is easiest to set these folders up from the beginning. We have the following typical layout for folders:

1. Project (e.g. Tau-MLi-2) 
    a. Figure (e.g. Figure 23) 
        i. Repeat (e.g. 1 or 2) 
          1. Cohort/Block (e.g. TM1) 
             a. QuPath (place for qupath project and exported 
             b. QVN (for QuickNII, Visualign, Nutil) 
                 i. Atlas 
                 ii. Input 
                 iii. Mask 
                 iv. Output_Left 
                 v. Output_Right 
    b. Figure (next one, same internal folder organization)… 


[STEPS]
TITLE: Liposome binding assay
AUTHORS: Christa Testerink, Anne Froyd-Rankenberg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Standard protocol to examine lipid binding of the proteins isolated from the E.coli with the GST tag</div></div>

[STEPS]
?. Mix lipids in chloroform in the right molecular ratios. Per sample 400 nmole (total) lipid is required. For (poly) phosphoinositides add MeOH (C:M 20:9)
?. Dry lipid mixtures for 30’ in the speed vac. Store at -20C
?. Add 500ul extrusion buffer per tube with lipid mixture. Vortex and let lipids rehydrate for 1-1.5 h at rt. Sonicate samples shortly (30’’)
?. Extrude the lipid suspension 13 x over polycarbonate membrane (pore size 0.2 m) to produce optically clear suspension of large unilamellar liposomes
?. Dilute liposome suspensions in three volumes (1,5ml) of binding buffer (x1) and pellet the liposomes by centrifugation at 20 300 rpm for 15’ at 22C (Sorval SM24 = 50 000g)
?. Resuspend liposome pellets in binding buffer (x1) to yield stock with final concentration of 16nM ( =400 nmole lipid per 25 ul of binding buffer)
?. Use 500 ng of protein per 400 nmol lipid. Dilute the protein in H2O until the volume is 20.8 ul. Add 4.2 ul 6x binding buffer to get a volume of 25 ul (but first spin the protein elution to get rid of protein aggregates or sepharose beads). If you are making master-mix of your protein samples – spin the master mix after adding the protein again in order to get rid of protein aggregates and other weird stuff.
?. Combine 25 ul of protein solution with 25 ul of lipid suspension. Incubate protein and liposomes for 45’ at room temperature on orbital shaker.
?. Centrifuge samples at 16 000 g for 30’
?. Harvest supernatant and wash the pellet once with 500 ul 1x binding buffer. Transfer resuspended pellet to the new tube (it will significantly improve cleaning of the pellet). Centrifuge pellet samples at 16 000 g for 30’.
?. Resuspend the pellet in 1x SB (33 ul) and add 4x SB to the supernatant fraction (16.5 ul). Incubate all samples at 95 C for 2’. Bring 10 ul of all samples on 10% SDS-gel for Coomassie Colloidal and / or Western Blot detection.
TITLE: Western blot analysis
AUTHORS: Kiichi Hirota

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

[STEPS]
?. [Cell lysis]
Scrape cells and transfer the suspension to a 1.5 ml microcentrifuge tube.
?. [Cell lysis]
Centrifuge at 10,000 x g for 5 minutes at 4°C and collect the supernatant in a new microcentrifuge tube.
?. [Cell lysis]
Determine protein concentration by using DC™ Protein Assay Kit.
?. [Separation of proteins by gel electrophoresis]
Load 35 µg of total protein on a SDS-PAGE gel.
?. [Separation of proteins by gel electrophoresis]
Run the gel for 1 h at 100 V.
?. [Semi-dry membrane transfer]
Transfer proteins onto a PVDF membrane using Trans-Blot Turbo™ Transfer System.
?. [Cell lysis]
Wash the cells with ice-cold PBS and add ice-cold RIPA buffer with cOmplete™ Protease Inhibitor Cocktail.
?. [Cell lysis]
Incubate the lysate on ice for 15 minutes.
?. [Western blotting]
Place the blot in Blocking One and incubate with agitation for 20 minutes.
?. [Western blotting]
Incubate the blot with appropriate dilutions of primary antibody at 4°C overnight.
?. [Western blotting]
Place the blot in TBST and wash three times, 5 minutes each.
?. [Western blotting]
Incubate the blot with the species-matched horseradish peroxidase-conjugated secondary antibody for 1 hour.
?. [Western blotting]
Place the blot in TBST and wash three times, 5 minutes each.
?. [Chemiluminescent Detection]
Add Chemi-Lumi One Super to cover the blot and incubate for 1 minute.
?. [Chemiluminescent Detection]
Detect chemiluminescence using LAS-4000mini image analyzer.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>End-stage renal disease (ESRD) is related to high morbidity, mortality, and impaired health-related quality of life. While hemodialysis (HD) is the current life-saving standard of treatment for patients with ESRD, their quality of life (QoL) remains far from desirable. Online HDF (OL-HDF), due to its convenience, could improve the QoL of patients with ESRD, however, this remains uncertain.</p>
<p>We will aim to assess the body of evidence of OL-HDF compared to HD regarding QoL in patients with ESRD.</p>
<p>We will comprehensively search in MEDLINE, EMBASE, The Cochrane Database of Systematic Reviews, and The Cochrane Central Register of Controlled Trials databases from their inception to February 2018. Reviewers working independently and in duplicate appraised the quality and included randomized controlled trials (RCTs) that evaluated, in patients with ESRD and HD or OL-HDF, QoL (Short Form Health Survey with 36 questions (SF-36) with physical component score (PCS) and mental component score (MCS) as well as scores about social activity, fatigue, and emotion). A meta-analysis of each outcome of interest will be performed using a random-effects model.</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how to make chemically competent <em>Vibrio natriegens</em> cells.</p>
<p>The protocol was described and published by Weinstock et al., 2016</p>

[BEFORE_START]
<p>Make sure you have all solutions there:</p>
<ul>
<li><strong>Brain heart infusion + V<sub>2</sub>-Salts</strong></li>
<li><strong>V2-Salts</strong>
<ul>
<li>NaCl 204mM (58.44g/mol) --&gt; 11.92176g for 200ml</li>
<li>KCl 4,2mM (74.55g/mol) --&gt;0,31311g for 200ml</li>
<li>MgCl<sub>2</sub> 23,14mM (203.3g/mol) --&gt; 7.704362g for 200ml</li>
</ul>
</li>
<li><strong>MgCl<sub>2</sub></strong> x 2H<sub>2</sub>O 100mM (203.3g/mol) --&gt; 0.10165g for 200ml</li>
<li><strong>CaCl<sub>2</sub></strong> x 2H<sub>2</sub>O 100mM (147.02g/mol) --&gt; 2.9404g for 200ml</li>
<li><strong>Modified Inoue buffer</strong>
<ul>
<li>MnCl<sub>2</sub> x 2H<sub>2</sub>O 55mM (161.87g/mol) --&gt; 1.78057g for 200ml</li>
<li>CaCl<sub>2</sub> x 2H<sub>2</sub>O 15mM (147.02g/mol) --&gt; 0.44106g for 200ml</li>
<li>KCl 250mM (75.55g/mol) --&gt; 3.7275g for 200ml</li>
<li>PIPES 10mM (302.37g/mol) --&gt; 4ml (from 0.5M stock solution) for 200ml</li>
</ul>
</li>
<li><strong>PIPES</strong> 500mM (302.37g/mol) --&gt; 7.55925g in 50ml (adjust pH 6.7)</li>
<li><strong>DMSO</strong></li>
</ul>

[GUIDELINES]
<p>All steps are done at room temperature (RT).</p>
<p>This protocol was published by Weinstock et al., 1016</p>

[STEPS]
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TITLE: Allende HRTEM study
AUTHORS: H. I. Cruz-Rosas, F. Riquelme, P. Santiago, L. Rendón, T. Buhse, F. Ortega-Gutiérrez, R. Borja-Urby, D. Mendoza, C. Gaona, P. Miramontes, G. Cocho.

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;"> </span><span style = "font-weight:bold;">SAMPLES.</span><span> We used a meteorite specimen of 297.8 g in weight, register number IG-A7, from the Colección Nacional de Meteoritas of the Instituto de Geología, Universidad Nacional Autónoma de México (UNAM), Mexico City (figure). For transmission electron microscopy (TEM), a ~ 4 cm long fragment of the large specimen were freshly fractured and cut by using a diamond knife in a clean room. Later, smaller clean fragments (~ 50 mg in total) were extracted from the interior, avoiding contamination from the fusion crust or surface areas. The resulting crude sample were powdered in an agate mortar and suspended in high-purity ethanol to achieve homogeneous dispersion without agglomerates. For HRTEM analysis, a 5µl pipette was add a portion of that suspension to a lacey carbon grid coated with formvar. Before being used, the TEM grid was carefully inspected in order to rule out any possible contamination.</span></div><div class = "text-block"><span style = "font-weight:bold;"> </span><span style = "font-weight:bold;">HRTEM.</span><span> High Resolution TEM imaging of chondrite samples was conducted using an Aberration Corrected Cold Field Emission Scanning Transmission Electron Microscope Jeol JEM-ARM200CF at the Centro de Nanociencias y Micro y Nanotecnologías, Instituto Politécnico Nacional, Mexico City. The TEM microscope is equipped with cold field emission gun, Cs-corrector, and high angle annular dark field (HAADF) detector and has ultra-high resolution of 0.72 Å. We utilized an electron beam spot with a condenser aperture of 60 nm at 200 kV for less than 30 seconds. Several locations on individual samples were analyzed. Fast Fourier Transform (FFT) analysis and image processing were applied using the freely available Digital Micrograph (GATAN) software attached at the microscope.</span></div></div>

[STEPS]
?.
TITLE: Nasal Lavage Sample collection and processing 
AUTHORS: maria.xylaki

[DESCRIPTION]
This protocol describes the nasal lavage fluid sampling that can be used for Omics (for Proteome analysis, see Schoenebeck et al, 2015; for Microbiome analysis see Heintz-Buschart et al, 2017) or RT-QuIC. Collection proceedure was adapted from B. Schoenebeck et al. 2015 (doi:10.1016/j.bbapap.2015.01.015).

[STEPS]
SECTION: Nasal Lavage Sample collection
1. Two nasal lavage samples (NLS) are collected per person in each step, one from each nostril.
Both nasal cavities are pre-washed with 2 mL of physiological NaCl solution with the use of a plastic disposable pipette to remove bacterial impurities.

SECTION: Nasal Lavage Sample collection
2. After 5 mins of incubation, each nasal cavity is rinsed again with 2 mL physiological NaCl solution with subsequent collection of the NLS into a tube.
SECTION: Nasal Lavage Sample collection
3. Following this step, an additional rinse is performed with 2 mL NaCl 10% (w/v) aqeous solution and collection of the NLS into a tube.
SECTION: Nasal Lavage Sample collection
4. A final rinse is performed with 2 mL NaCl 20% (w/v) aqeous solution and collection of the NLS into a tube.
SECTION: Nasal Lavage Sample collection
5. Depending on downstream applications, NLS samples can be immediatelly frozen at -80 °C  until analysis or centrifuged at 10.000 x g for 5 mins at 4 °C in order to remove cellullar debri and then stored.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for add azide modified nucleotide to 3' end of unmodified DNA olgionucleotide using terminal transferase. Protocol adapted from <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4787804/" target="_blank" rel="noopener noreferrer">Winz et al. 2015</a>. </p>

[STEPS]
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TITLE: Mitochondrial Bioenergetics
AUTHORS: mitsjoecohen  

[DESCRIPTION]
Mitochondrial bioenergetics plays a key role in a variety of basic cellular processes, such as energy production, nucleotide biosynthesis, and metabolism. Not only the basic system of cell production and death (apoptosis), it is also necessary for embryonic development. Besides, mitochondrial bioenergetics is involved in the adaptive process and its gene products in species, as well as in the formation of animal reproductive disorders.


[STEPS]
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Enzymatic Assay of Trypsin Inhibition
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol has been standardized for analysis of protease inhibitors in soybean leaf extract, but can be easily adjusted for other biological samples.</div></div>

[STEPS]
?. [Prepare the solutions and the workspace]
Preparation of solutionsTrypsin solution:Dilute 1.25 mg of bovine trypsin in 1 mL of water pH 3.0 (adjust with HCl).L-BApNA stock solution:Dilute 130.47 mg L-BApNA in 5 mL DMSO (concentration: 6.0 10-2 M). Store at -20 °C and protected from light.L-BApNA usage solution (freshly prepared):Dilute 200 mL of stock solution in 10 mL of 100 mM Tris-HCl buffer, pH 8.2 and 20 mM CaCl2. Protect from light.
?. [Prepare the solutions and the workspace]
Separate three microtubes and name them "blank", "control (uninhibited test)" and "test (inhibited test)".Pipette the following reagents.Blank: 500 µL BApNA usage solution and 500 µL buffer.Control: 100 μL trypsin solution and 400 μL buffer.Test: 100 µL enzyme, 100 µL leaf extract (source of inhibitors) and 300 µL buffer.
?. [Prepare the solutions and the workspace]
Mix the three microtubes by inversion and equilibrate to 25°C for 5 minZero spectrophotometer with blank content at 410 nm
?. [Prepare the solutions and the workspace]
To the control microtube, add 500 µL of the BApNA usage solutionImmediately mix by inversion and mark the time and pour the contents into a cuvette. After 30 s of reaction onset, monitor readings at 410 nm for 120 s
?. [Prepare the solutions and the workspace]
Add 500 µL of the BApNA usage solution to the test tubeImmediately mix by inversion and mark the time and pour the contents into a cuvette. After 30 s of reaction onset, monitor readings at 410 nm for 120 s
?. [Prepare the solutions and the workspace]
Calculations‰ Inhibition = (ΔA410 nmcontrol - ΔA410 nmtest) * 100 / (ΔA410 nmcontrol - A410 nmblank)orTrypsin Inhibitor Units / mL = (ΔA410 nmcontrol - ΔA410 nmtest) / (8800 * time * leaf extract volume)A410 nmblank = Absorbance in the blank at 410 nmΔA410 nmcontrol = Absorbance variation in the control sample at 410 nm within 120 secondsΔA410 nmtest = Absorbance variation in the test sample at 410 nm within 120 seconds8800 = extinction coefficient of p-nitroanilide at 410 nmtime = 120 secondsleaf extract volume = Volume of inhibitor source used (in milliliters)The presence of trypsin inhibitors in the leaf extract decreases the enzymatic activity and this inhibition can be represented in the Lineweaver-Burk graph, where, as the inhibitor concentration increases, the slope of the line also increases.