eamag/protocols-with-steps huggingface dataset:
TITLE: FloodLAMP Inactivation Solution Prep v1.0
AUTHORS: Randy True

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Inactivation Solution includes TCEP, EDTA, NaOH, and water. It should be made in a chemical fume hood, as the TCEP is caustic. It is very straightforward to make if starting with liquid-form TCEP. We have not made it with powder-form TCEP, which is about 30% cheaper at low volume retail prices from Sigma. </div><div class = "text-block">We have yet to do long-term stability studies; however, we think it's fine stored at room temperature for at least a few weeks. If yellow Binding Solution does not go clear upon addition to an inactivated sample or PBS+Inactivation Soln, suspect that the Inactivation Solution may have degraded, and repeat with another aliquot or freshly-made Inactivation Solution.</div><div class = "text-block">It has also been suggested by Albert Yu of Brandeis has recommended using a 1.4N NaOH concentration for saliva instead of 1.15N NaOH. NaOH should be scale up by 1.4/1.15 and water down for this. </div></div>

[STEPS]
?. [Preparation]
Add all TCEP to tube, break glass vial tops off, using the 1000uL pipette extract the liquid with the vial inverted
?. [Preparation]
Get amounts of TCEP, EDTA, NaOH and Ultrapure at volumes shown in chart below:
?. [Preparation]
Add EDTA
?. [Preparation]
Vortex for 10s, be sure the tube is fully closed
?. [Preparation]
Add NaOH
?. [Preparation]
Vortex for 10s, be sure the tube is fully closed
?. [Preparation]
Add UltraPure dH2O
?. [Preparation]
Vortex for 10s, be sure the tube is fully closed
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a protocol for transfection of H4 (Human Brain Neuroglioma) in either 10% or 100% FBS using FuGENE HD. </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>In recent years, fruit flies (Drosophilidae) in cherry trees in northern China have become an increasingly serious problem. In this study, we examined the species composition and dynamics of fruit flies in cherry orchards by lure traps placed  at different heights, and in three different cherry cultivars from April through September, between 2014-2016 in Qingdao, Shandong Province. The total number of fruit flies increased year by year. Four species were captured: <em>Drosophila melanogaster </em>Meigen, <em>D</em><em>.</em><em> suzukii </em>Matsumura, <em>D</em><em>.</em><em> hydei </em>Sturtevant,<em> </em>and <em>D</em><em>.</em><em> immigrans</em> Sturtevant. The dominant species was<em> D. melanogaster, </em>being 66.7% of the total, followed by<em> D. suzukii </em>Matsumura (29.7%). <em>D. hydei </em>and <em>D. immigrans </em>accounted for 3.0 and 0.6%, and for these last two species, no peak period of abundance was observed. In general, drosophilid fruit flies appeeraed in April, and peaked around mid-July. Over the three years of the study, peak fruit fly abundance was later each year, being at the end of June in 2014, and in the middle of July in 2015 and 2016. There were significant differences in the number of<em> Drosophila</em> species among cherry cultivars as well asin the number of <em>Drosophila</em> trapped at different heights. The optimal height for trap placement was at 1m above ground level, which is recommended as a standard height for monitoring trap placement.</p>

[STEPS]
TITLE: Lysis Aspergillus niger, extracting and purifying DNA
AUTHORS: Laure Leynaud-Kieffer

[STEPS]
?. [Grow the Biomass]
-4 days before the lysis, inoculate on MMA (Minimal Media Agar - see Transformation protocol for MMA recipe) at 30oC  the strain that you want to analyze.
?. [Harvest the spores]
-Once enough biomass grew on the slants, you are ready to do the lysis-Make sure that before you use the entire slant for the lysis, back up your strain by harvesting some spore into a new slant (30oC for 4 days). -Add 1mL of 0.2% tween-20 into the slant and vortex to have the spores into the solution. You don't need super concentrated spores to make the lysis worked.
?. [Extracting the DNA]
-Add 20 uL of mycelia, into 480 uL of the 1st lysis buffer (400 mM of Tris-HCl pH 8.0, 60 mM of ethylene diaminetetraacetic acid (EDTA) pH 8.0, 150 mM NaCl and 1% (v/v) sodium dodecyl sulfate (SDS)). The total volume is 500 uL, mix and incubate for 10 minutes at room temperature. -After incubation add 100 uL of the 2nd buffer (2 M potassium acetate, and 7.6% glacial acetic at pH 4.8), the solution turns white opaque. Centrifuge at 10,000 rpm for 2 minutes.-Right after the centrifuge, 500uL of the supernatant is transferred to a new 1.5-mL Eppendorf tube and centrifuged again for 2 minutes at 10,000 rpm.
?. [Purifying the DNA]
-Right after centrifuge, 400 uL of the supernatant is transferred to a new 1.5-mL Eppendorf tube and washed with 400 uL of isopropyl alcohol (>99%). Invert the tube couple of times before centrifuge for another 2 min at 10,000 rpm. - Right after centrifuge discard the supernatant and rinse the rest of the DNA (contain at the bottom of the tube) with 300 uL 70% EtOH. Invert the tube couple of times before centrifuge for another 2 min at 10,000 rpm. -Right after centrifuge discard the supernatant and evaporate the rest of the EtOH using a rotavapor (30C, Alcohol mode,  1h15min). -After all the alcohol has been evaporated, the DNA is dilute in 50 uL of dH2O, and ready for PCR (LongAmp Taq Master Mix- 1uL of DNA for 25 uL of reaction).
TITLE: Localised axotomy of human Cortical Neurons (CNs) from induced pluripotent stem cells (iPSCs)
AUTHORS: Quyen Do, Federico Nebuloni, Richard Wade-Martins

[DESCRIPTION]
This protocol describes the process followed to perform localised axotomy of iPSC-derived human cortical axons cultured within fluid-walled dumbbells in 6 cm TCT-treated Petri dishes. A similar protocol was first described by Soitu et al., 2020 to perform wounds assays in monolayer cell cultures. 

[STEPS]
SECTION: Localised axotomy assay
1. On Day 20, remove the FC40 overlay from the Petri dish where human Cortical Neurons (CNs) were cultured inside fluid-walled dumbbells.
SECTION: Localised axotomy assay
2. Gently add ~2 ml of coating-prep medium (see Materials) to destroy every fluid wall without peeling cells off the substrate. 
SECTION: Localised axotomy assay
3. Remove medium and wash with PBS twice. Be careful with cells peeling. 
SECTION: Localised axotomy assay
4. After last wash, add ~5 ml of fresh medium (Neurobasal supplemented with B27). 
 

SECTION: Localised axotomy assay
5. Equip the fluid printer with a 1 mL glass syringe filled with medium.
SECTION: Localised axotomy assay
6. Place dish into the fluid printer. 
SECTION: Localised axotomy assay
7. Fluid printer automatically perform axotomy by means of a submerged medium jet. Such jet is held at a fixed height above cells and it is moved around the dish by the printer traverse to cross perpendicularly across axon bundles at the midpoint. 

Key parameters:
jet height =300 μm
jet flow rate = 480 μl/min
traverse speed = 960 mm/min
 

SECTION: Localised axotomy assay
8. Remove medium and overlay fresh FC40. 
SECTION: Localised axotomy assay
9. New fluid-walled dumbbells can be fabricated around axotomized cultures following steps 1.5 and 1.6 in Protocol: Fabrication of fluid-walled dumbbells and generation of the human corticostriatal pathway
SECTION: Live-imaging of axonal regeneration
10. On DIV 0, fluorescent live images of all dumbbell conduits were taken just prior to CNs replating to serve as initial normalising timepoint. 

SECTION: Live-imaging of axonal regeneration
12. Medium was changed every other day from now on for the next 20 days.
SECTION: Live-imaging of axonal regeneration
11. Images were taken on a digital SLR camera (Nikon D7100 DSLR) connected to an epi-fluorescence microscope (Olympus IX53; 1.25×, 4×, 10×, 25× objectives) equipped with a translation stage and an overhead illuminator (Olympus IX3 with filters). 
SECTION: Live-imaging of axonal regeneration
13. Similar live images were taken again on day 20 prior to axotomy, and subsequently 36, 60, and 84 hours post-axotomy.
TITLE: MAP-2 &amp; GFAP Staining Protocol
AUTHORS: Machlusil Husna, Kusworini Handono, Hidayat Sujuti, Aulanni'am, Ettie Rukmigarsari

[DESCRIPTION]
Neurodegeneration due to neurotoxicity is one of the phenomena in temporal lobe epilepsy. Experimentally, hippocampal excitotoxicity process can occur due to kainic acid exposure, especially in the CA3 area. Neuronal death, astrocyte reactivity and increased calcium also occur in hippocampal excitotoxicity, but few studies have investigated immediate effect after kainic acid exposure. The organotypic hippocampal slice culture (OHSC) is a useful model for studying the neurodegeneration process, but there are still many protocol differences. In this study, minor modifications were made in the OHSC protocol

[STEPS]
SECTION: Sample Preparation
1. -Tissue slices were fixed using 4% PFA for 15 minutes
-Wash with PBS for 3 times (5 minutes each)
-Remove PBS and add 0,1% Triton X-100 for 30 minutes
-Wash with PBS for 3 times (5 minutes each)
-Add 10% BSA for 30 minutes at room temperature
SECTION: Assay Procedure
2. -Incubate with primary antibody MAP-2 (1:500) and FDAP (1:500) overnight at temperature 4 oC. 
-Wash with PBS for 3 times (5 minutes each)
-Incubate with secondary antibody for 1 h at room temperature
    - Goat anti-Rabbit IgG-F (1:500) for MAP-2 (Santa Cruz Biotechnology sc-53805, Lot#K1109)
    - Anti-mouse Rhodamine (1:500) for GFAP (Rockland, paint : 610-1002)
-Wash with PBS for 3 times (5 minutes each)
-Observe using CLSM with magnification of 100X and a wavelength of 488 nm (MAP-2) and 543 nm (GFAP)
TITLE: Homogenate of A. cervicornis
AUTHORS: Stephanie Rosales

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is to study disease in </span><span style = "font-style:italic;">Acropora cervicornis</span><span> by using a disease homogenate method. These methods are adapted from methods presented in Muller 2018 </span><a href="https://elifesciences.org/articles/35066" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://elifesciences.org/articles/35066</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. Collect fragments of A. cervicornis showing signs of active white-band disease. These fragments are preferably collected from the same area and with similar disease progression.
?. Hold diseased fragment over Ziploc bag (** this will require two people**) and remove tissue by air brushing.Per fragment, remove ~10 cm2  of live tissue from the diseased fragment by airbrushing off the tissue within 5 cm of the advancing band with 0.2 microns of filter-sterilized seawater.
?. With a serological pipette apply 100 mL of homogenate above each coral fragment in the randomly selected disease experimental tanks.
?. Repeat steps 1-5 with healthy coral samples and add healthy coral homogenate to the control tanks.
?. Sterile razor with bleach and RNA removal reagent then collect coral tissue near the base of the fragment.
?. Mix disease homogenate in the bag.
?. Save at least 500 ul of homogenate for sequencing.
?. Place tissue in ZR BashingBeadTM Lysis Tubes (0.1 & 0.5 mm) with DNA/RNA shield (lyses cells, inactivates nucleases and infectious agents and is ideal for safe sample storage and transport at ambient temperatures)
TITLE: RNAPath Target Identification via Fluorescent Hybridization
AUTHORS: Devin Willis

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

[STEPS]
?. [Sample Preperation]
Suspend RNA pellet in
[TE Buffer]
?. [Hybridization]
Add  to the RNA suspension
[Molecular Instruments COVID Probe Set]
?. [Hybridization]
Place Molecular Instruments Alexa Flour 488 B1 HCR Amplifier in  for
90 °C
?. [Hybridization]
Remove Molecular Instruments Alexa Flour 488 B1 HCR Amplifiers and place at  for
0 Room temperature
?. [Hybridization]
Add   of each Molecular Instruments Alexa Flour 488 B1 HCR Amplifier H1 and H2 to the RNA suspension
2 µl
?. [Hybridization]
Dilute   of 20x SSC Buffer to 5x SSC Buffer
100 µl
?. [Hybridization]
Add   of 5x SSC Buffer to the RNA Suspension
4 µl
?. [Hybridization]
Incubate at   for
37 °C
?. [Imaging]
Remove sample from incubation and pipette  onto a glass slide with a coverslip
1 µl
?. [Imaging]
Image slide under a fluorescent microscope with filter cubes for FITC or Alexa Flour 488
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the library preparation for Illumina sequencing. It accompanies the <em>GigaScience</em> publication:</p>
<p> </p>
<p>Benjamin Istace, et al. (2017) De novo assembly and population genomic survey of natural yeast isolates with the Oxford Nanopore MinION sequencer. <em>GigaScience...</em></p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Luminex Milliplex Cytokine/Chemokine 22-plex MAG manufacturer's protocol.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides an efficient and standardized way to prepare samples ready for mass spec analysis.</p>

[BEFORE_START]
<p>Prepare solutions as described in the steps.</p>

[STEPS]
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TITLE: UC Davis - Erectile Dysfunction
AUTHORS: Li-Ming Jin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Erectile function is well regulated by vasorelaxant and vasoconstrictive mechanisms, which can be perturbed by various disease states including cardiovascular disease, diabetes, and hormonal insufficiencies. This protocol enables us to asses erectile function by measuring the mean arterial blood pressure (MAP) and intracavernosal pressure (ICP) upon electrical stimulation of the cavernous nerve.</div><div class = "text-block"><span style = "font-weight:bold;">Modified from:</span><span> Burnett et al Science. 1992 Jul 17;257(5068):401-3.; Quinlan et al J Urol. 1989 Mar;141(3):656-61.; and Jin et al J Sex Med. 2008 Mar;5(3):544-51.</span></div></div>

[STEPS]
?. Rats or mice are anesthetized with an intraperitoneal injection with 50 mg pentobarbital/kg weight.
?. A midline incision was made from umbilicus to pubis.
?. The penis was denuded of skin, and the prepuce circumcised. The testes and epididymides were repositioned into the abdomen after they were divided from their scrotal attachments.
?. Bipolar silver wire electrodes were attached unilaterally to the cavernous nerve that arises from the ipsilateral pelvic plexus situated dorsolateral to the prostate.
?. Intracavernous pressures were measured (Gould Polygraph, Cleveland, Ohio) with a 25-gauge needle inserted unilaterally at the base of the penis and connected to a pressure transducer.
?. Penile erection was induced electrically with a Grass S48 square wave stimulator at 1-5 volts, 16 htz for 1 min.
?. Neurostimulation was performed until a 10s maximal pressure recording was achieved, but no stimulation lasted longer than 90s. At least 3 min elapsed between repeated stimulations.
?. Maximum intracavernosal pressure (Max ICP) and total area under the ICP curve above baseline were normalized by systemic mean arterial blood pressure (MAP) and used as indices of erectile function.
TITLE: Manual Aseptic Sort Procedure for BD FACS Aria II/III/Fusion
AUTHORS: Jamie C Tijerina

[DESCRIPTION]
This is an alternative to the automated aseptic sort procedure designed to decontaminate the lines of the BD FACS Aria Fusion instrument. Bypasses filters and other internal components that may be damaged by 10% bleach or FACSClean. This protocol is useful if the connections used for the aspetic sort on the wetcart have failed and the procedure needs to be performed before a service call can be placed. 

[BEFORE_START]
Autoclave or chemically decontaminate a spare stainless steel tank for your instrument before starting. Perform as many tasks as possible within a biosafety cabinet.

[STEPS]
1. Perform decontamination of tank (see SOP). The preference is to use a spare 10L main tank when performing this procedure. 
2. Fill the tank with 4L of 10% bleach.
3. Connect the air line and the sheath line directly to the tank. Do not connect the filter as it cannot withstand bleach.
4. Place an already damaged nozzle designated specifically for cleaning procedures (not the closed loop or nozzles to be used during sort) on the instrument. Even though it is a cleaning nozzle that should have only had cleaning solutions run through it, sonicate the nozzle for 1 minute before use as aggregates can still form and block the orifice.
5. Turn on the stream. Run the stream of bleach for 20 minutes.
6. After 20 minutes, turn off the stream.
7. Disconnect the tank, empty it into the sink. Rinse the tank well with DI water 2x.
8. Fill the tank with 4L of DI water directly from dispenser.
9. Connect the air line and the sheath line directly to the tank. Do not connect the filter.
10. Turn on the stream. Run the stream of DI water for 20 minutes.
11. After 20 minutes, turn off the stream.
12. Disconnect the cleaning tank.
13. Place a freshly decontaminated tank with sterilized sheath onto the wetcart. Connect the air line and the sheath line (with or without filter depending on the experiment protocol) and perform a regular fluidics startup using the closed loop nozzle. Or, if the instrument will be shut down, perform a regular fluidics shutdown.
TITLE: Canine Enteric Virus Detection Assays
AUTHORS: Come J Thieulent, Mariano Carossino, Laura Peak, Udeni B. R. Balasuriya

[DESCRIPTION]
The Canine Enteric Virus Detection Assays is intended as an in vitro veterinary reagent set, based on Reverse Transcription quantitative PCR (RT-qPCR), for the detection of canine adenovirus type 1 (CAdV-1), canine distemper virus (CDV), canine enteric coronavirus (CECoV), canine parvovirus (CPV), canine rotavirus A (CRV) and SARS-CoV-2 in rectal swabs and fecal samples.

[GUIDELINES]
SHIPPING and STORAGE:

The Canine Enteric Virus Detection Assays are shipped on dry ice. Reagents should arrive frozen. The Reagents in the purple and red tubes may arrive liquid, this will not result in a reduction in performance.

All reagents should be stored at -20°C upon arrival. All reagents can be stored for a minimum of one year (from the date of shipment) at -20°C without showing a reduction in performance. Positive controls should be stored at -80°C.

LIMITATIONS:
Strict compliance with the instructions is required for optimal results.
Appropriate specimen collection, transport, storage, and processing procedures are required for the optimal performance of this test.
The presence of RT-PCR inhibitors may cause false negatives.
Results of Canine Enteric Viruses Detection Assays need to be interpreted in consideration of all clinical and laboratory findings.

QUALITY CONTROL:
The specificity of each test was validated using a panel of reference and related canine respiratory pathogens.
The analytical sensitivity of each assay was determined using ten-fold dilution of in vitro transcribed RNA or plasmid copies number. All assays have a limit of detection (LOD95)  15 copies/l.

[STEPS]
SECTION: Reaction Set Up
1. Thaw all reagents on ice.


SECTION: Reaction Set Up
2. Centrifuge all reagents on a benchtop centrifuge to ensure no liquid is in cap and keep on ice 
 

SECTION: Reaction Set Up
3. Setup the Master Mix according to the following table 1:

  

 
 
 

SECTION: PROGRAMMING THE THERMOCYCLER
4. Select the following fluorescence channels: ABYTM, FAMTM, JUNTM and VICTM.
 

SECTION: PROGRAMMING THE THERMOCYCLER
5. The standard mode should be selected, following the table below:
  

SECTION: RESULTS INTERPRETATION
6. Before analysis of results, the threshold value of each fluorescent dye must be manually set in the region of exponential amplification, typically 0.1 × ΔRn value at the plateau phase.
SECTION: RESULTS INTERPRETATION
7. Each assay is considered valid if the following criteria are met:
  

SECTION: RESULTS INTERPRETATION
8. The results are qualitative (Positive or Negative). A specimen is considered positive if the Ct value obtained is below the following Ct cut-off values:
TITLE: Bone Marrow Derived Macrophage (BMDM) differentiation and maintenance
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This protocol describes the isolation of bone marrow derived macrophages.

[STEPS]
1. 
 
Euthanize mice via CO2 or isoflurane inhalation and cervical dislocation.
2. Collect femurs and flush cavities with 5 mL ice-cold PBS.

3. Then, collect bone marrow cells by centrifugation 0.3 rcf, 5 min.

4. Resuspend the resulting pellet and differentiate for 6 days in culture media containing: DMEMF12 supplemented with 20% FBS, 20% L929 conditioned media, 1% penicillin-streptomycin and 1% GlutaMAXTM.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Long-read sequencing (e.g. Nanopore's MinION or PacBio) has the potential to dramatically improve genome assembly but the quality of the reads is critically dependent upon the quality of the input gDNA. <br /><br />Here I describe the extraction of high molecular weight DNA from the calcareous sponge,<em> Sycon capricorn</em>. I choose to use Qiagen RLT buffer in the initial lysis step as this solution results in higher molecular weight DNA compared to alternatives. I suspect that RLT is deactivating endogenous DNases that would otherwise digest the gDNA.</p>

[BEFORE_START]
<p>Equilibrate phenol:chloroform:isoamyl alcohol to pH 8.0 and allow enough time (several hours) for phases to separate.<br /><br />Chill absolute and 70% ethanol at 4°C.<br /><br />Fill the 20 mL syringe with vacuum grease and autoclave.</p>

[STEPS]
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