eamag/protocols-with-steps huggingface dataset:
TITLE: “O-map/way method-site”: A sampling method for complete plant site inventories in large forests in the moderate/colline zone using orienteering maps or maps of similar quality
AUTHORS: André Strauss

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">“O-map/way method-site”: A sampling method for complete plant site inventories in large forests in the moderate/colline zone using orienteering maps or maps of similar quality </span><span></span></div><div class = "text-block">By André Strauss, August 29. 2019</div></div>

[STEPS]
?. Making 3 complete inspection rounds in the season, when the given plant species can be best recognized, in the same year, ideally carried out by different persons, in the forest target area using an orienteering map or a map of similar quality (in paper or in an electronic device; ideally at a scale of 1: 5 000) with additional means, such as GPS, compass and recording devices, so that every accessible place on the target area is screened for new plant sites.
?. 
?. 
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?. Screening in a first step for sites along all accessible ways in the target area, recording localisation of sites by GPS (and optionally by sketching in the orienteering map) and noting the plant site area sizes and the abundance of plants within the site and optionally their state. The maximum distance from the way for recording can be e.g. 3 m for herbaceous plants, 10 m for at least 1 m high shrubs and 20 m for at least 2 m high trees and all species which can clearly be recognized from the way. Sketching in the inspection track in the orienteering map.
?. Screening in a second step for new sites by meandering in 5-20 m broad loops in the accessible subareas between all ways (not including sites already recorded by screening along ways) and other lines visible on the orienteering map and/or in nature, so that every accessible place can be searched for sites. Recording of sites and additional notes are done as given above for sites along ways.
?. This sampling method can possibly also be applied to complete plant site inventories in large forests in other zones using orienteering maps or maps of similar quality.
TITLE: Native Nanobleach of native nanodiscs
AUTHORS: Caroline Brown, Gerard Walker, Snehasish Ghosh, Kallol Gupta, Moitrayee Bhattacharyya

[DESCRIPTION]
This is a protocol to conduct Native Nanobleach of native nanodiscs.

[STEPS]
1. Note, this method is adapted from Walker, Brown, et al. (DOI: 10.1038/s41565-023-01547-4)

2. Prepare flow chamber by treating with poly-L-lysine (PLL) PEG and PEG-biotin.
3. Add streptavidin to flow chamber.

4. Add biotinylated GFP-nanobody to flow chamber.
5. Add native nanodiscs to the flow chamber for immobilization.
6. Conduct step-photobleaching analysis using TIRF microscopy.
TITLE: How to Design a Primer
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes how to design a primer. To see the full abstract and additional resources, visit </span><a href="https://www.addgene.org/protocols/primer-design" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/primer-design/</span></a><span style = ":;">.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol aims to assess:</p>
<p>- the interaction between two proteins harboring two parts of the gaussia luciferase (Glu1 and Glu2)</p>
<p>- in presence of a third protein co-expressed</p>
<p>- in presence of a rabies virus</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Introduction: This study was conducted to describe the repeat multiparametric MRI (mpMRI)<br />changes occurring in prostate cancer (PCa) patients during active surveillance (AS), and to study<br />possible associations between mpMRI-related parameters in predicting prostate biopsy (Bx)<br />Gleason score (GS) upgrading &gt;3+3 (GU) and protocol-based treatment change (TC).<br /><br />Materials and methods: The study cohort consisted of 76 AS patients with GS 3+3 PCa<br />and at least two consecutive mpMRIs of the prostate performed between 2006 and 2015. Patients<br />were followed according to the Prostate Cancer Research International Active Surveillance (PRIAS) protocol and additional mpMRIs. The primary end points were GS upgrading (GU) (&gt;3+3) in protocol-based biopsies and protocol-based TC.<br /><br />Results: 53/76 (69%) patients had progression (PIRADS upgrade, size increase or new<br />lesion[s]), 18/76 (24%) had radiologically stable disease, and 5/76 (7%) had regression<br />(PIRADS or size decrease, disappearance of lesion[s]) in repeat mpMRIs during AS. PIRADS<br />scores 4 to 5 in the initial mpMRI was associated with GU (p=0.008) and protocol-based TC<br />(p=0.009). Tumour progression on repeat mpMRIs was associated with TC (p=0.045) but not with<br />GU (p=1.00). PIRADS scores 4-5 predict GU (sensitivity 0.80 [95% confidence interval (CI);<br />0.51-0.95, specificity 0.62 [95% CI; 0.52-0.77]) with PPV and NPV values of 0.34 (95% CI;<br />0.21-0.55) and 0.93 (95% CI; 0.80-0.98), respectively.<br /><br />Conclusion: mpMRI is a useful tool not only to select but also to monitor PCa<br />patients on AS.</p>

[STEPS]
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TITLE: Chromosomal DNA extraction from Gram-positive bacteria, V3
AUTHORS: Anders Kiledal, Julia A Maresca

[DESCRIPTION]
Extraction of high-molecular-weight DNA from Gram-positive bacterial species, with optional steps for removing surfactants. This DNA is suitable for sequencing and the protocol can be scaled up at least 5-fold. Modified from a protocol by Tina Wecke, LMU-Munich. 

[BEFORE_START]
Grow culture to high cell density and prepare all solutions. 

[GUIDELINES]
Recommend wearing gloves throughout and working in a biosafety cabinet if possible to prevent contamination. This protocol can be scaled up at least 5-fold. If the isopropanol precipitation step is used, subsequent steps do not have to be scaled up unless the culture volume is substantially larger.

[STEPS]
SECTION: Grow culture
1. Inoculate 10 mL rich medium from a fresh overnight culture, and incubate at appropriate temperature on shaker. At OD600 of ~0.8-1.0, harvest cells by centrifugation (10 min., 5000 rpm).
SECTION: Cell lysis
2. Resuspend cell pellet in 2 mL TEN (10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 150 mM NaCl).
SECTION: Cell lysis
3. Add 100 μL lysozyme (20 mg/mL) and incubate for 20 min at 37ºC.
SECTION: Cell lysis
4. Add 20 μL RNAse (10 mg/mL) and incubate for 3 min at 65ºC.
SECTION: Remove surfactants (optional)
6. IF THE STRAIN PRODUCES A SURFACTANT THAT INTERFERES WITH THE PHASE SEPARATION, Add 0.1 volume 3 M sodium acetate and 1 volume cold isopropanol, mix, and incubate on ice for 20 min. Centrifuge for 10 min at 5000 rpm and decant the supernatant. Then resuspend in 400 μL TEN and 550 μL TEN* and transfer to a microcentrifuge tube.
SECTION: Phenol &amp; chloroform:isoamyl alcohol extractions
7. Add 900 μL phenol, mix by inversion. Centrifuge for 5 min. at 13000 rpm and transfer the upper phase to a clean microcentrifuge tube.
SECTION: Phenol &amp; chloroform:isoamyl alcohol extractions
8. Re-extract once with phenol (1 volume) and twice with chloroform: isoamyl alcohol (24:1 v/v, 1 volume)
SECTION: DNA precipitation
10. Collect DNA by coiling on the end of a glass Pasteur pipet.
SECTION: DNA precipitation
11. Air dry, then resuspend DNA in 100 μL sterile water overnight at 4ºC.
SECTION: Cell lysis
5. Add 40 µl SDS, a small scoop of proteinase K and 550 µl TEN*. Vortex, then incubate at 60°C for 2 hours.
SECTION: DNA precipitation
9. Transfer upper phase to 10 mL cold 100% ethanol.
TITLE: TAP agar plate preparation
AUTHORS: Joao Vitor Molino

[STEPS]
?. Always follow aseptic techniquesAutoclavate freshly prepared TAP media with   with a magnetic bar insideMix the flask using a magnetic stirrer to evenly dissolve the agar in the solutionFor antibiotic containing plates, cool down the flask temperarute until    -    or until it is possible to hold it without hurting the skin. Add antibiotics in the desired amount, and mix using a magnetic stirrer to evenly dissolve the antibioticPour the molten media in sterile petri dishs. (Keep volume constant among plates).Wait for media to solidifyFor antibiotic containing plates, cool down the flask temperarute until   -   or until it is possible to hold it without hurting the skin. Add antibiotics in the desired amount, and mix using a magnetic stirrer to evenly dissolve the antibioticPour the molten media in sterile petri dishs. (Keep volume constant among plates).Wait for media to solidifyStock at -  until use. (Storage for 2 months was used without noticeable difference)
[bacteriological agar]
55 °C
60 °C
4 °C
8 °C
TITLE: Immunohistochemical analysis of ganglion neurons innervating the lower urinary tract [keast-001-stage03]
AUTHORS: Janet Keast, Peregrine Osborne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for studies of neurons containing retrograde tracer dye in adult rat dorsal root ganglia (DRG) or pelvic ganglia (PG; synonym, major pelvic ganglia). Cryosections of ganglia are mounted directly on slides for immunohistochemical staining. Antibodies have been selected to distinguish different neurochemical classes of PG neurons or to specifically identify subclasses of sensory neurons that are myelinated (immunoreactive for NF200 [neurofilament, 200 kD]) or nociceptive (immunoreactive for TRPV1). </div></div>

[STEPS]
?. [Preparation of cryosections]
Cryoprotect tissues in phosphate-buffered saline (PBS; 0.1 M, pH7.2) containing 30% sucrose. This should be performed at 4C, 24-72h prior to cutting.
?. [Preparation of cryosections]
Embed tissues in cryomold using OCT, freeze in cryostat and cut sections (14 µm), distributing sections progressively across sets of 3 slides so that each slide has non-consecutive sections.
Chrome-alum treated slides are pre-coated (subbed) with 1% gelatin, dried and stored at room temperature. This slide treatment enhances section adhesion while retaining good surface tension of the antibody droplets.
?. [Immunostaining]
Air-dry slides at room temperature for at least 10 minutes.
?. [Immunostaining]
Wash sections in PBS (10 min).
?. [Immunostaining]
Incubate sections in blocking solution at room temperature in a humidified dark chamber for 1-2 h.
?. [Immunostaining]
Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 18-24h in a humidified dark chamber. Antibodies are diluted in hypertonic PBS.
?. [Immunostaining]
Wash sections in PBS (15 min)
?. [Immunostaining]
Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) for 2-3h in a humidified dark chamber. Antibodies are diluted in hypertonic PBS.
?. [Immunostaining]
Wash sections in PBS (15 min)
?. [Immunostaining]
Coverslip in preferred anti-fade mountant.
?. [Microscopy]
Retrogradely labeled neurons are counted and classified according to their immunoreactivity, including only nucleated neuronal profiles in the analysis.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>MATERIALS:</strong> <br /><br />1) 60-65°C heat block or water bath <br />2) Microfuge <br />3) 1.5 and 2.0 mL microfuge tubes (screw-cap) <br />4) 50 mM Tris-HCl, pH 7.5, 10 mM MgCl<sub>2</sub> <br />5) 10% NP-40 or Triton X-100 <br />6) DNAse I, 2.0 mg/mL in 50 mM Tris-HCl, pH 8.0. <br />       Store in aliquots at -20°C. Do not refreeze unused material, discard. <br />7) Proteinase K, 2.0 mg/mL in 50 mM Tris-HCl, pH 8.0. <br />       Solution can be thawed and frozen several times, but should be discarded after that. <br />8) 10% Na sarcosyl <br />9) CHCl<sub>3</sub>:Isoamyl alcohol (24:1) <br />10) 500 mM EDTA, pH 8.0 <br />11) 3 M NaOAc <br />12) 100% EtOH <br />13) Tris buffer-saturated phenol <br />14) 10 mM Tris-HCl, pH 8.0, 1 mM EDTA (1X TE)

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Loess (decayed limestone) topsoil based soil and water media concentrate for use with RO/DI water. NOT for use with treated tap or spring water! Intended for use in filamentous algae, volvox and euglenoid culture.</p>

[BEFORE_START]
<p>Collect 5 to 10kg of loess soil. Break up any large lumps. Sift out roots and other large debris with a #8 seive (0.93 in/2.362mm) USE A STAINLESS STEEL SEIVE! Copper and Brass seives may contaminate the medium making it inhospitable to algae! Allow to air dry.</p>

[GUIDELINES]
<p>This protocol is for fabrication of a concetrated media stock. It should not be used 'straight' for growing organisms.</p>
<p>Note that this method utilizes locally available loess soil. Results vary with other types of soil.</p>
<p> </p>
<p>This media requires an overnight cooling and settling period. It is best to start it in the afternoon, and finish it next morning.</p>

[STEPS]
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TITLE: Improving pneumonia diagnoses using pulse oximetry at rural health institutions in southern Ethiopia: Protocol for a cluster-randomized controlled trial
AUTHORS: Solomon Hailemariam1, 2, 3*, Yabibal Gebeyehu4, Eskinder Loha1, 5, Kjell Arne Johansson2, Bernt Lindtjørn1, 2

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Abstract </span></div><div class = "text-block"><span style = "font-weight:bold;">Background</span><span>: Hypoxemia is common in children with pneumonia. The World Health Organization and United Nations Children’s Fund developed the Integrated Management of Childhood Illness strategy for diagnosing pneumonia. But the strategy has limitations to accurately detect hypoxemia in children with severe pneumonia. This study will assess whether pulse oximetry improves health workers’ performance in diagnosing severe childhood pneumonia and whether the pulse oximeter is a cost-effective tool to improve diagnoses of severe pneumonia at primary health care units in South Ethiopia.</span></div><div class = "text-block"><span style = "font-weight:bold;">Methods</span><span>: A cluster-randomized controlled trial will be conducted at 24 health centres in southern Ethiopia from September 2018 to February 2019. The trial has two arms: the first arm will examine the combined use of the Integrated Management of Childhood Illness (IMCI) strategy with pulse oximetry, and the second arm will examine the use of the IMCI strategy alone. The study population will include children between 2 months and 59 months of age who present with cough or difficult breathing. The primary outcome of this trial is severe childhood pneumonia, as diagnosed via the IMCI guideline and pulse oximetry. Secondary outcomes will be referred cases of severe pneumonia, and treatment failure on day 14 after enrolment. A cost effectiveness study will be conducted simultaneously.</span></div><div class = "text-block"><span style = "font-weight:bold;">Discussion</span><span>: This study will determine if using a pulse oximeter at health facilities in rural Southern Ethiopia improves diagnostic accuracy for paediatric severe pneumonia. Our secondary aim will be to evaluate if the benefit of pulse oximetry justifies its cost. </span></div><div class = "text-block"><span style = "font-weight:bold;">Trial registration: </span><span>PACTR201807164196402(14/06/2018). URL:</span><a href="https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=3466" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=3466</span></a><span>. </span></div></div>

[STEPS]
TITLE: Preparation of LRRK2 RCKW trimer cryo-EM grids
AUTHORS: Mariusz Matyszewski

[DESCRIPTION]
This protocol has been adapted from Deniston et al (https://doi.org/10.1038/s41586-020-2673-2)

Original protocol by Colin Deniston. Adapted to protocol.io by Mariusz Matyszewski.

This protocol describes how to create cryo-EM grids for LRRK2 RCKW. In particular, this protocol was used to obtain a high-resolution cryo-EM structure of the LRRK2 RCKW trimer, as well as lower resolution structures of RCKW monomers and dimers.

[GUIDELINES]
Newer protocol is available (see updated protocol here) with better results for monomeric protein. This is included mainly for archival reasons.

[STEPS]
SECTION: Preparing Sample
1. Dialyze purified LRRK2 RCKW into the final LRRK2 buffer (see Materials)
SECTION: Preparing Sample
2. Dilute the protein to the desired final concentration.

 

SECTION: Freezing Grids
3. Glow discharge grids.
We used UltrAuFoil Holey Gold 1.2/1.3 300 mesh grids and glow discharged them at 20 mA for 20 s in a K100 instrument.

SECTION: Preparing Sample
2.1. (Optional) If adding inhibitors, add them after diluting to the final concentration and incubate on ice for 60 min . If using Apo protein, then proceed immediately.
SECTION: Freezing Grids
4. Apply protein to grids and plunge freeze.
We used a Vitrobot (FEI) to blot away excess sample and plunge freeze
SECTION: Freezing Grids
5. Store grids in liquid nitrogen until ready for imaging.
TITLE: 10x Genomics Multiome ATAC+GEX 
AUTHORS: Andrew Houston

[DESCRIPTION]
Link to 10x Genomics Multiome protocol to assess chromatin accessibility and gene expression from a dissociated sample

[STEPS]
1. Protocol can be found here:
https://cdn.10xgenomics.com/image/upload/v1666737555/support-documents/CG000338_ChromiumNextGEM_Multiome_ATAC_GEX_User_Guide_RevF.pdf

Transposition through cDNA pgs. 28-57
ATAC library construction pgs. 49-52
GEX library construction pgs. 59-67
TITLE: siRNA-mediated knockdown of TFE3
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol details siRNA-mediated knockdown of TFE3.

[STEPS]
SECTION: siRNA-mediated knockdown of TFE3
1. On the day before transfection, plate 100,000 cells per well in a 6 well dish.
SECTION: siRNA-mediated knockdown of TFE3
2. Change media 60 min before transfection (2 mL with no antibiotics).
SECTION: siRNA-mediated knockdown of TFE3
3. The next day, assemble the siRNA transfection reaction by combining 7.5 µL of 20 micromolar (µM) siRNA, 5 µL RNAiMAX transfection reagent (Invitrogen), and 500 µL Opti-MEM (Invitrogen) in a sterile 1.7ml tube.
SECTION: siRNA-mediated knockdown of TFE3
4. Incubate transfection mix at Room temperature for 30 min.

SECTION: siRNA-mediated knockdown of TFE3
5. After incubation, add the transfection mixture drop wise to the cells.
SECTION: siRNA-mediated knockdown of TFE3
6. Collect the lysates for immunoblotting 2 days post-transfection.
TITLE: SOP88v1_TGD_Lentivirus transduction in EndoC-bH1
AUTHORS: Yunkyeong Lee

[DESCRIPTION]
For lentivirus transduction in EndoC-bH1 cells

[STEPS]
1. After generating lentivirus (Virus core facility), keep them at -80C. 
2. Seed 4x10^6 cells in T25 flask or 7x10^6 cells in T75 flask (EndoC-bH1 cells). 
3. The next day, calculate MOI and left the appropriate amount of lentivirus (MOI must be tested to ensure appropriate before transduction) on the cells for 8 days. 
4. Check whether GFP is detected in the cells using fluorescence microscope if GFP is included in the construct (In my experience, the signals became stronger over time, so I checked it everyday, and Day 8 was the best. I think it is related we split EndoC-bH1 cells once a week (cell doubling time)).
5. 8 days later, split them into separate plates.
6. CAUTION: 
1. There is no significant difference depending on whether polybrene is present or not.
2. When puromycin was used, the cells did not grow well, thus it was not possible to make cell stocks or any experiments. Additionally, since GFP is present in almost all cells after viral (containing GFP) transduction, selection by puromycin is unlikely to be necessary.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">This protocol describes the procedure to obtain single bacterial colonies composed of multiple sectors of different strains. Each sector is originated from different founder cells (situated in close proximity to each other at the moment of plating). This protocol is based on Hallatschek et al. 2007 (</span>Hallatschek O, Hersen P, Ramanathan S, Nelson DR. Genetic drift at expanding frontiers promotes gene segregation. Proc Natl Acad Sci U S A. 2007 Dec 11;104(50):19926-30).</p>
<p> </p>
<p>This assay has been used for low cost and open source fluorescence imaging (please see https://osf.io/dy6p2/ for further information &amp; data, https://github.com/SynBioUC/FluoPi for code and http://docubricks.com/viewer.jsp? for hardware assembly)</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div style="left: 279.287px; top: 275.557px; font-size: 15.4212px; font-family: serif; transform: scaleX(1.02067); text-align: left;" data-canvas-width="54.1293365688">This protocol is designed to allow you to clean large numbers of 15 mL vials to the highest standards of cleanliness, but in a time-efficient manner.</div>

[STEPS]
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TITLE: Chemically competent cells transformation
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Chemically competent cells transformation protocol</div></div>

[STEPS]
?. [Preparation for competent cells]
Inoculate a single colony into 5 mL appropriate media containing appropriate antibiotics
?. [Preparation for competent cells]
Incubate overnight using appropriate temperature and shaking conditions
?. [Preparation for competent cells]
Dilute overnight culture to an optical density of 0.01 in 10 mL of appropriate media containing appropriate antibiotics
?. [Preparation for competent cells]
Incubate at appropriate temperature and shaking conditions until optical density reaches 0.4 to 0.6. Do not allow to grow past this point.
?. [Preparation for competent cells]
Spin down cells at 3500 RPM at 4°C for 10 min
?. [Preparation for competent cells]
Resuspend pellet in 10 mL ice-cold 0.1M CaCl2
?. [Preparation for competent cells]
Place cells on ice for 20 min (do not go under this time; can go longer)
?. [Preparation for competent cells]
Spin down cells as in step 5.
?. [Preparation for competent cells]
Resuspend pellet in 1 mL ice-cold 0.1M CaCl2 + 13% glycerol (for a higher concentration of cells, use less)
?. [Preparation for competent cells]
Place on ice for 20 min
?. [Preparation for competent cells]
Use immediately or store at -80°C (0.25 mL/tube)
?. [Transformation]
Add 50 µL competent cells into a sterile Eppendorf tube
?. [Transformation]
Add 1 µL plasmid DNA or ligation reaction (can vary this)
?. [Transformation]
Place on ice for 30 min
?. [Transformation]
Heat shock at 42°C for 1 min
?. [Transformation]
Place on ice 2 min
?. [Transformation]
Add 0.5 mL recovery media (SOC, LB, etc.)
?. [Transformation]
Incubate for 1 hour at appropriate growth conditions.
?. [Transformation]
Plate 200 µL on appropriate plate with appropriate antibiotics.
?. [Transformation]
Incubate at 37°C overnight