eamag/protocols-with-steps huggingface dataset:
TITLE: Beetle Cleaning
AUTHORS: Jiri Hulcr, Demian F Gomez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes surface-sterilization or surface-cleaing techniques for the beetles before the microbe isolation.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol is a modified version of the Qiagen RNeasy assay for isolating total RNA from Heterosigma akashiwo. We used this protocol to obtain high quality RNA for downstream analysis such as quantitative PCR. </p>
<p> </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the protocol for the Q5&reg; Site-Directed Mutagenesis Kit without competent cells

[STEPS]
?. 
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TITLE: Chloral Hydrate Seed Clearing
AUTHORS: Gabrielle Sandstedt, Andrea Sweigart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">To characterize early seed development in Mimulus (1-5 days after pollination), we clear seeds with chloral hydrate and quickly obtain images of embryo and endosperm development using a Differential Interference Contrast Microscope. </div></div>

[STEPS]
?. [Dissecting fruit and clearing]
Emasculate a bud from some maternal plant. 2 to 3 days later pollinate by selfing/outcrossing or use an unfertilized fruit.
?. [Dissecting fruit and clearing]
Remove the developing fruit 1 to 5 days after pollination or 2 to 3 days after emasculation. In Mimulus, this protocol is useful for capturing early seed development (0- to 5 days). After 5 days, the seed tissue thickens and becomes difficult for viewing.
?. [Dissecting fruit and clearing]
Pipette  of diluted Hoyer's solution onto a glass slide and dissect developing ovules from the fruit directly onto the glass slide using sharp forceps
10 µl
?. [Dissecting fruit and clearing]
After dissection, pipette about  of the diluted Hoyer's over the developing ovules and place a coverslip on top. Then, set the slide flat, upright in a 4ºC fridge.
30 µl
?. [Dissecting fruit and clearing]
Depending on how developed the ovules are, clear for at least 1 to 12 hours before viewing with a Differential Interference Contrast Microscope.
?. [Prepare Hoyer's Solution]
Hoyer's Solution:  gum Arabic, glycerol,  Chloral Hydrate,  Water.  Diluted Hoyer's solution:  Hoyer’s solution:  10% gum Arabic
19 g
12 g
250 g
75 ml
10 ml
7.5 ml
2.5 ml
TITLE: Via Keto Apple Gummies Australia Reviews- Price or Chemist Warehouse Buy
AUTHORS: health  

[DESCRIPTION]
Via Keto Apple Gummies - Natural products are always a better choice due to their medicinal potential and guaranteed safety.

[STEPS]
1. Via Keto Apple Gummies is a weight loss component primarily based on a ketogenic food regimen that includes essential nutrients together with omega-3s, multivitamins, as well as protein to offer speedy weight management effects without facet effects. Official Web: https://www.mynewsdesk.com/nealthnewscart/pressreleases/via-keto-apple-gummies-australia-reviews-chemist-warehouse-and-keto-bhb-formula-2022-3192503
TITLE: CollectingCitationsfromText
AUTHORS: Rebecca Hedreen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Basic steps and scripts used for translating text citations to bibtex files suitable for loading into citation management software or citation analysis scripts. 3 publically available webpage mounted scripts are suggested, that require no programming.</div></div>

[STEPS]
?. [Preparing text file]
Copy citations from the source document(s) into a text (.txt) document.
?. [Preparing text file]
Edit the text document so that each citation is on a separate line with one blank line between each citation. Not all the scripts require a blank line between citations, but it does improve readability and importing.
A text editor that includes line numbers (the type used by software programmers) makes this step easier.
?. [Processing citations]
I've found 3 web-mounted scripts that will do text file to bibtex file translation.Anystyle.io can be used on the web, or as a Ruby script. This is the most flexible script and it allows detailed editing before the final file export. The website is privately hosted, so it's not always been available.This PERL script is mounted on a website and will do basic translation. No support, but it's quite good.Hosted by the University of Toronto. Requires registration. Basic editing and checking in Google Scholar is available. Accurate and reliable.All 3 will export a .bib file suitable for importing into citation managers.
?. [Export]
Once the script has exported a .bib file, load it into your citation manager of choice. Zotero has been the most reliable for importing.
Zotero also produces more standardized .bib and .ris files than the scripts, especially the older scripts. I have imported into Zotero, exported a bib file, and imported into other software if a direct import didn't work.
TITLE: fastANI analysis protocol
AUTHORS: Jamie Harrison, David J Studholme

[DESCRIPTION]
this is the protocol to conduct ani analysis between groups of genomes using fastANI and produce the heatmap figure in R using the pheatmap package

[STEPS]
SECTION: create directories
1.2. mkdir analysis query_genomes reference_genomes
 

SECTION: create directories
1.3. move to query dir


 

SECTION: create directories
1.1. 


 
 
SECTION: create directories
1.4. 
 

SECTION: create directories
1.5. 
 

1. Create directory structure and link files
SECTION: create directories
1.8. move to reference dir


 

SECTION: create directories
1.9. 
 

SECTION: create directories
1.10. 
 

SECTION: create directories
1.12. 
 

SECTION: create directories
1.6. check soft links and remove bad links

 

SECTION: create directories
1.11. 
 

SECTION: create directories
1.7. 
 

SECTION: create directories
1.13. 
 


SECTION: create directories
1.14. 
 

SECTION: create directories
2. 
 

SECTION: create directories
3. 
 

SECTION: create directories
3.1. 
 

SECTION: create directories
4. 
produce heatmap figure in R

SECTION: create directories
4.1. 
 

SECTION: create directories
4.2.
TITLE: Microbiological contamination of young children’s hands in rural Bangladesh: Associations with child age and observed hand cleanliness as proxy
AUTHORS: Sarker Masud Parvez, Rashidul Azad, Amy J. Pickering, Laura H Kwong, Benjamin F. Arnold, Musarrat Jabeen Rahman, Md. Zahidur Rahman, Mahfuja Alam, Debashis Sen, Sharmin Islam, Mahbubur Rahman, John M. Colford, Jr, Stephen P. Luby, Leanne Unicomb, Ayse Ercumen

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for collecting gas for measurement of carbon isotope descrimination during photosynthesis.</p>
<p> </p>
<p>See the following references for more background:</p>
<p>H. Griffiths (1993) <a href="https://link.springer.com/book/10.1007/978-94-011-1566-7" target="_blank" rel="noopener noreferrer">Photosynthesis and Production in a Changing Environment</a> pp 181-1927</p>
<p>Farquhar et al.  (1989) Annu. Rev. Plant Physiol. Plant Mol. Bioi. 1989. 40:503-37 </p>

[STEPS]
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TITLE: CIDC_S16_LC_MS_Celegans_Extraction_Protocol
AUTHORS: Brianna M Garcia, Carter  Asef

[DESCRIPTION]
A sample preparation protocol for lyophlized C. elegans samples to be analyzed via LC-MSMS

[STEPS]
SECTION: Homogenization
1. Samples are removed from -80 °C

SECTION: Homogenization
2. (3) 2.0mm zirconium oxide beads and ~ 75 µL  volume  of 0.5mm glass beads are added to each sample tube.

SECTION: Homogenization
3. Samples are placed in Tissuelyser II using adapter trays chilled at -80C and homogenized at 1800rpm for 3 min. 

SECTION: Homogenization
4. Samples are now homogenized.
SECTION: Extraction
5. 750 µLof 100% isopropanol is added to the 1.5mL Eppendorf tube containing the homogenized sample. The sample tube is lightly vortexed to create a suspension of homogenized sample and the resulting slurry is transferred to a new 2.0mL Eppendorf tube, leaving the beads behind in the original tube. This step is repeated so that a total of 1.5 mL of solvent is transferred to the new 2.0mL Eppendorf tube.

SECTION: Extraction
7. Samples are placed in the centrifuge and spun at max speed (22100G) for 5 min
SECTION: Extraction
8. Supernatant of each sample is transferred to a new 2.0 mL Eppendorf labeled for RP chromatography and dried down using steps 14 through 18

SECTION: Extraction
9. The second round of the sequential extraction is 1.5 mL of 80/20 methanol/water per added directly to the pellet remaining after centrifugation.

SECTION: Extraction
10. Samples are shaken using the Fisher Scientific Isotemp High Speed Shaker at 1500rpm for 30 min

SECTION: Extraction
11. 2.0mL Eppendorfs are placed in the centrifuge and spun at max speed (22100G) for5 min

SECTION: Extraction
12. Supernatant of each sample is transferred to a new 2.0 mL Eppendorf labeled for HILIC chromatography and dried down using steps 14 through 18

SECTION: Sample drying/storage
14. Samples are placed in a Labconco CentriVap concentrator and monitored until they have completely dried (roughly 4-5 hours)
SECTION: Sample drying/storage
15. Once dry, samples are stored at -80 °C until they are to be run on the LC-MS instrument.

SECTION: Sample drying/storage
18. Transfer to LC-MS vial
SECTION: Sample drying/storage
16. When preparing samples to run on LC-MS, reconstitute samples with 75 µL of 100% isopropanol for reverse phase and 80/20 methanol/water for HILIC.

SECTION: Sample drying/storage
19. After LC-MS analysis, samples can be stored at -80C until ready for IMS analysis
SECTION: Extraction
6. Samples are vortexed for 1 min and then extracted over night  at -20C

SECTION: Extraction
13. Pellets are dried for 1 hour and stored at -80 °C 

SECTION: Sample drying/storage
17. Vortex for 1 minute, then centrifuge at max speed (22100G) for5 min
TITLE: Artificial Cerebrospinal Fluid I (ACSF.I)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to prepare Artificial Cerebrospinal Fluid I (ACSF.I). ACSF.I is used for applications including transcardial perfusion prior to fresh mouse brain slicing and as a bathing solution during fresh mouse brain slicing. </div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Human Tissue Slicing and Dissections for Nuclear Isolations
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the slicing and microdissection procedure on frozen post-mortem human brain tissue to be used to isolate nuclei for subsequent transcriptional profiling.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U01MH114812. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Stereotaxic viral injections and array implantation
AUTHORS: Mai-Anh Vu, mwhowe

[DESCRIPTION]
We have developed a new micro-fiber array approach capable of chronically measuring and optogenetically manipulating local dynamics across over 100 targeted locations simultaneously in head-fixed and freely moving mice, enabling investigation of cell-type and neurotransmitter-specific signals over arbitrary 3-D volumes . This protocol includes the stereotaxic viral injections and array implantation steps. Please contact us (mwhowe@bu.edu) if you are interested in using this technique. 

[STEPS]
SECTION: viral injection
1. Mice were anesthetized under isoflurane (1-3%) and placed in a stereotaxic frame. 
SECTION: viral injection
3. Mice were injected with AAVs to express genetically encoded proteins for optical measurements and manipulations. Virus (~200-800nL per site) was injected stereotaxically through a pulled glass pipette (diameter 30-50µm) at a rate of ~100nL/min.
SECTION: viral injection
3.1. For striatum expression, virus was injected at 10-40 total locations chosen to maximize overlap with fiber positions.
SECTION: viral injection
3.2. For midbrain targeting, for one set of mice, we injected the 4 sites relative to bregma: AP = -3.05, ML = 0.6, DV = -4.6 and DV = -4.25; AP = -3.5, ML = 1.25, DV = -3.9 and DV = -4.5.
SECTION: viral injection
3.3. For a midbrain targeting in another set of mice, we injected at 3.07mm caudal to the bregma at 4 sites: ML = 0.5mm, DV =-4.00 mm and -4.25 mm, ML = 1mm, DV = -4.125mm and ML = 1.5 mm, DV = -3.8 mm below the dura.
SECTION: array implantation
4. The fiber array was then mounted in the stereotaxic and slowly lowered into position until one side contacted the skull surface. 
SECTION: array implantation
5. A thin layer of Kwik-Sil (WPI) was applied to seal off the exposed edges of the craniotomy, and then Metabond (Parkell) was used to secure the plastic frame to the skull.
SECTION: array implantation
6. After this initial layer of Metabond solidified, a metal headplate and ring(Atlas Tool and Die Works, as in Howe and Dombeck, 2016; Dombeck et al., 2010) were secured to the skull with Metabond and the surface covered with a layer of blackened Metabond (carbon powder, Sigma).
SECTION: array implantation
7. Finally, a small cylindrical plastic protective cap cut to extend just above the fiber bundle end was secured around the bundle and covered on the inside surface with blackened Metabond.
SECTION: viral injection
2. A large craniotomy was performed with a surgical drill (Midwest Tradition 790044, Avtec Dental RMWT) to expose the cortical surface above the striatum.
SECTION: array implantation
4.1. Precise targeting of the array and rigid placement into the stereotaxic is essential to avoid mis-placement of fibers (which may result in tissue distortion) and tissue damage caused by lateral movements of the fibers.
TITLE: Single-cell single-unit recordings in vitro
AUTHORS: Nicola Biagio Mercuri

[DESCRIPTION]
Single units in slices

[STEPS]
SECTION: Single-cell single-unit recordings
1. The slice is placed in a submerged recording chamber in continuously flowing ACSF (34°C) saturated with O2/CO2 gas mixture (95/5%).
SECTION: Single-cell single-unit recordings
1.1. ACSF composition (in mM): 126 NaCl, 2.5 KCl,1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 10 glucose and 25 NaHCO3.
SECTION: Single-cell single-unit recordings
2. A glass microelectrode filled with ACSF (tip resistance 5–10 MΩ) is slowly deepened into the slice, with voltage signals captured through an Axoclamp 2A amplifier using Clampex 10 software and digitized with a
Digidata 1440 (Molecular Devices, LLC., 3860 N First Street San Jose, CA 95134 USA).
SECTION: Single-cell single-unit recordings
3. When fast spontaneous transients are observed, the depth of the recording electrode is adjusted to obtain voltage signals as high as possible
TITLE: Chemogenetic modulation of catecholaminergic neurons
AUTHORS: Cristian González-Cabrera, Csilla Novák, Maria P. Contreras, Ernesto Durán, Matthias Prigge

[DESCRIPTION]
This protocol outlines a method for modulating noradrenergic and dopaminergic neuronal activity in the LC and SNc-VTA, respectively, using Cre-dependent hM3D and hM4D expression. It details stereotactic injection techniques for these chemogenetic tools and the preparation of the J60 agonist for intraperitoneal delivery. Developed for longitudinal studies spanning up to four weeks, the protocol aims to assess neuromelanin's activity-dependent accumulation through daily agonist administration.

[STEPS]
SECTION: Stereotactic injection
1. Stereotactic injection of viruses into DbH-Cre (JAX# 033953)  or DAT-Cre (JAX# 006660) animals

Viral Construct:
pAAV-hSyn-DIO-hM4D(Gi)-mCherry (Addgene link) 
Volume: 400ul
pAAV-hSyn-DIO-hM3D(Gq)-mCherry (Addgene link)
Volume: 400ul

(in our experiment we mix our DIO-hTyr Virus 1:1 with DREADDs)

Steroetactic coordinates:
Subtantia nigra

ML: 1.4mm AP: 3.25mm DV: 4.0mm (blunt needle / NF34BL-2)

Locus coeruleus

ML: 1.2mm AP: 5.2mm DV: 3.5mm (blunt needle / WPI NF34BL-2)
ML: 1.5mm AP: 5.2mm DV: 4.2mm (beveled needle / WPI NF34BV-2)

Injection set-up:
Hamiliton syringe couple t oa WPI injector (UMP3T-1) (link) 
Injection speed 100nl/min

SECTION: Preparation agonist solution
3. Preparation of agonist solution 

Several agonists are available for chemogenetic modulation with advantages and disadvantages, i.e., clozapine N-oxid (CNO), clozapine, or JP60. We are using J60 (JHU37160)
Please look at the reference for more detail (Bonaventura et al., 2019)

 

Solve entire vial (1mg) in 10ml saline (0.9% NaCl) 
     (Compound can be ordered from Torcris (link))

     The solubility is very good, so by vortexing for a couple of seconds it should be already dissolved.

Aliquot in a volume of 0.3ml and freeze at -20degree

DO NOT USE IT FOR MORE THAN 1 MONTH!


SECTION: Expression
2. Assess accepted pain management protocol for injected animals, Start experiment as earlyas 3 weeks post-injections
SECTION: Modulation of Neuronal Activity
4. Animals are injected at a dosage of 1mg/kg body weight. Therefore, each aliquot is the maximum for one animal. The injections are intraperitoneal, as shown in the picture. When administering repeated injections, use different locations in the belly to avoid the same injection site.
TITLE: MicroCT protocols for scanning of embryos and juvenile Hexaplex trunculus
AUTHORS: Eva Chatzinikolaou, Kleoniki Keklikoglou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Micro-computed tomography (micro-CT) is a high-resolution 3D-imaging  technique which is now increasingly applied in biological studies  focusing on taxonomy and functional morphology. The creation of virtual  representations of specimens can increase availability of otherwise  underexploited and inaccessible samples. This protocol aims to standardise micro-CT scanning procedures for embryos and juveniles of the marine gastropod species </span><span style = "font-style:italic;">Hexaplex trunculus. </span></div></div>

[STEPS]
?. [Sample preparation]
Anesthetization of embryos and juvenile gastropods with 7% MgCl2.
?. [Sample preparation]
Sample placement inside a plastic white pipette tip without any scanning medium (in the air).
?. [microCT scanning]
?. [microCT scanning]
Scanning parameters for embryos and juvenile Hexaplex trunculusVoltage: 59 kVCurrent :167 μAFilter: none Pixel size: 2 μmCamera binning: 1 × 1Exposure time: 325 ms for embryos and 316 ms for juvenilesRotation: 360° for embryos and 180° for juvenilesRotation step: 0.20º for embryos and 0.25° for juvenilesFrame averaging: 3 for embryos and 5 for juveniles
?. [Images reconstruction]
Projection images were reconstructed into cross sections using the SkyScan’s NRecon software (Bruker, Kontich, Belgium)
?. [Images reconstruction]
Upload projection images
?. [Images reconstruction]
Perform X-Y alignment
?. [Images reconstruction]
Reconstruction parametersSmoothing: 2Ring artifact correction: 20Beam hardening correction: 59%Attenuation coefficients: 0 - 1.127 for embryos and 0 - 1 for juvenilesSave as: 16-bit TIFF images
?. [Sample preparation]
Removal of embryos of Hexaplex trunculus from their egg capsules.
TITLE: IVF Bioscience Bovine Slaughterhouse Protocol
AUTHORS: Jake Silcock

[STEPS]
?. [Aspiration and IVM]
Dish and Media Preparation
?. [Aspiration and IVM]
Add  of BO-IVM medium per well to 4-well plates without an Oil overlay and place in the incubator
500 µl
?. [Aspiration and IVM]
Additionally, prepare one 35 millimetre dish with  of BO-IVM per 4-well plate and place in the incubator without an Oil overlay
3 ml
?. [Aspiration and IVM]
Aspiration
?. [Aspiration and IVM]
Warm the saline solution to
32 °C
?. [Aspiration and IVM]
Take note of the arrival time and temperature of the ovaries
?. [Aspiration and IVM]
Wash the ovaries in the warmed saline and hold in a thermo-container of warmed saline
?. [Aspiration and IVM]
Add  of heparin to the 50 ml tube prior to aspiration
140 µl
?. [Aspiration and IVM]
Aspirate all follicles between 2-15 millimetres using an 18-guage needle connected to the pump system. The opening of the needle should face downwards
?. [Aspiration and IVM]
Aspirate no more than  of follicular fluid per tube and note the number of ovaries aspirated on the lid of the tube
20 ml
?. [Aspiration and IVM]
Prepare and equilibrate IVM wells at   in 6% carbon dioxide in a humidified atmospheric air (21% oxygen) for 02:00:00  before use
38.8 °C
?. [Aspiration and IVM]
Preheat the Wash medium to
36 °C
?. [Aspiration and IVM]
Set the heating plate to   and connect the vacuum equipment
34 °C
?. [Aspiration and IVM]
Collection and Maturation
?. [Aspiration and IVM]
Use 9 cm Petri dishes marked with a square grid pattern
?. [Aspiration and IVM]
Prepare one dish per tube of follicular fluid. Mark the dish with the corresponding tube number.
?. [Aspiration and IVM]
Add  of Wash to each dish
7 ml
?. [Aspiration and IVM]
Prepare three 35 millimetre dishes with  of Wash in each. Mark these dishes as 1, 2 and 3
2.5 ml
?. [Aspiration and IVM]
Transfer the oocyte pellet from the bottom of the 50 ml tube into the larger petri dish using a plastic transfer pipette
?. [Aspiration and IVM]
Search through the dish systematically and transfer all oocytes to the smaller dish marked 1
?. [Aspiration and IVM]
Repeat the transferring of the oocyte pellet and search, twice for each tube
?. [Aspiration and IVM]
Once all oocytes have been collected, wash through dish 2 and then 3. Count the number of collected oocytes
?. [Aspiration and IVM]
Rinse once through the 35 millimetre dish containing BO-IVM
?. [Aspiration and IVM]
Once rinsed, transfer the oocytes to the 4-well plate containing BO-IVM. Do not transfer more than 45 oocytes per  well.
500 µl
?. [Aspiration and IVM]
Incubate at  and 6% carbon dioxide in humidified atmospheric air (21% oxygen) for between  and .
38.8 °C