eamag/protocols-with-steps huggingface dataset:
TITLE: Neurolucida 360: Detecting muscle fiber orientation 
AUTHORS: Maci Heal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Detecting muscle fiber orientation in 3D microscopy images using Neurolucida 360.</div></div>

[STEPS]
?. [Setup]
Launch SPARC-enabled Neurolucida 360.
?. [Using the Detect Orientation tool]
When your image is loaded, close the 3D window. From the TRACE ribbon, select the Detect Orientation icon.
?. [Using the Detect Orientation tool]
?. [Using the Detect Orientation tool]
NOTE:  If you are unsure of the size of your object of interest, find the measure line tool from the TRACE ribbon.
?. [Using the Detect Orientation tool]
Once your object size is specified, select Detect Orientation. The detection algorithm will then detect the orientation of the local region of image signal at that designated size.A progress indicator will display while Neurolucida 360software determines muscle fiber orientation.
?. [Using the Detect Orientation tool]
View results in the 3D window. The muscle fiber orientations are modeled with red arrows overlaid on the image data.
?. [Setup]
Open a microscopy image via the Open icon, File>Open, or dragging and dropping into the program window.NOTE: MBF Bioscience software supports a variety of image file formats from common microscopy vendors such as JPX/JP2, TIFF, LIF, ND2, IMS, OIF/OIB, almost all CZI, and more.
?. [Setup]
The SPARC Vocabulary Services window will appear. The dialog is displayed so that you can specify subject-specific metadata and gain access to the SciCrunch database to retrieve anatomical terminology lists compliant with FAIR data principles to use during annotation.
?. [Setup]
Fill out the Subject Information and select your Criteria for Anatomic Terms. Then select Begin.
?. [Using the Detect Orientation tool]
Save the data file in XML format. The orientation data for all detected vectors will be written to the XML file for use in your individual analysis pipelines and/or MAP-Core segmentation registrations to organ scaffolds.
?. [Using the Detect Orientation tool]
The algorithm detects the orientation of image regions based on a user-defined size (orientation scale). The orientation score for each detected vector, represented as an arrow is calculated; values range from 0 to 1, with 1 representing perfect alignment in a principal direction. Only the fiber direction vectors are displayed in Neurolucida 360 software, however, the orientation of the sheet vector (lateral direction normal to the fiber in the plane of the sheet) and the vector normal to these two are recorded in the XML data file.
TITLE: S-3 SOIL STORAGE
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol describes soil storage.

[GUIDELINES]
OBJECTIVE

To outline steps for properly storing field-collected soil samples and nucleic acid samples purified from these soil.

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on storage of soil samples and their purified nucleic acid to preserve their integrity for downstream nucleic acid extraction and/or sequencing library preparation.


MAINTENANCE OF EQUIPMENT

Decontaminate a PCR workstation by keeping the UV light on for 15 min.
 
 


[STEPS]
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
1. 
 

Cool 96-well microfuge tube racks on ice.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
2. Each collected soil sample will be transferred to a 50 mL falcon tube with a unique sample ID. Put falcon tubes into cryo/freezer box.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
3. Label the boxes with unique box ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
4. Close the box tightly, secure with clear Saran wrap and immediately transfer to -80 °C freezer.

SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
5. Update the freezer inventory so that samples can be tracked properly.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
6. 
 
In the clean PCR workstation, carefully transfer the eluted total nucleic acid to a 96-well PCR microplate, make sure to keep samples in the exact same locations corresponding to the rack where the original ticks were stored.
 

SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
7. Cover the 96-well PCR microplate with adhesive film to prevent spill over or contamination.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
8. Label the film with a unique plate ID.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
9. Immediately transfer the 96-well PCR microplate to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
10. Update the freezer inventory so that samples can be tracked properly.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p> To perform a comparative analysis of the physiological, biochemical and agronomical parameters of durum wheat cultivars (<em>Triticum durum</em> Desf.) under water-deficit conditions, five cultivars were grown under irrigated (control) and rainfall (stressed) conditions. Different agro-physiological and biochemical parameters were studied: electrolyte leakage, relative water content, chlorophyll fluorescence, proline, soluble sugars and specific peroxidase activity</p>

[STEPS]
TITLE: UC Davis - Digestible Energy by Bomb Calorimetry
AUTHORS: Trina Knotts

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Changes in fecal energy loss can result in differences in net energy balance and feed efficiency, and may result from, e.g., alterations in bile acid production, secretion or re-uptake, and changes in activities of enzymes involved in macronutrient digestion or uptake (e.g., pancreatic lipase).  This assay will be coupled to food intake determinations and will involve fecal collection and feces weight measures for 48h, which can be coordinated with the meal pattern studies (catalog item D4003) or indirect calorimetry (catalog item D4007).  Diet and fecal energy content will be measured by bomb calorimetry and the following reported:  fecal energy density, total fecal energy loss/day, and fecal energy loss as a percent of energy intake.   Alternatively, fecal samples and diet samples can be examined without food intake measurements.  In this case, only diet and fecal energy density will be reported.  Additional tests can be done through proximate analysis (D4005- Digestible Energy by Proximate Analysis) to determine protein or fat absorption.</div></div>

[STEPS]
?. Mice are singly housed in clean cages with fresh food and water and low bedding.
?. Weigh mouse and “food-in” (grams of rodent diet provided at start of experiment).
?. Save several pellets of rodent diet for analysis (e.g., for energy or macronutrient content)-Store at 4C in airtight container.
?. At 24h, weigh mouse and “food –out” (grams of rodent diet remaining)
?. At the end of 48h collection period, weigh mouse and “food-out”.
?. Swap out the cage bottoms with new clean bottoms, replace feeder insert/water bottle and return animals to housing system.
?. For each cage, carefully inspect and remove each piece of bedding using tweezers to collect all feces.   Inspect each pellet of feces to assure that there is no spilled food or fibers of hair or bedding attached.  If significant food spillage is found in cage bottom, weigh in pre-tared weigh boat to add back to food measurement.
?. Place feces in labeled pre-weighed 1.5 ml tube.
?. Weigh tubes and record weight.
?. Subtract this value from original tube weight to determine fecal “wet” weight.
?. After collection, fecal samples are stored at -80C until analysis for bomb calorimetry.
?. Data collected:48h food intake 48h fecal “wet” weight fecal energy density total fecal energy loss/day fecal energy loss as a percent of energy intake breakdown of fecal energy loss:  protein, fat, carbohydrateEnclose fecal/diet sample log with samples and send on dry ice to:Mouse Biology Program C/O UC Davis MMPC University of California 2795 Second Street, Suite 400 Davis, CA 95618 530-757-3333 Please include MBP/MMPC project # in reference section on package documentation and send the FedEx/UPS tracking number information to: mmpc-bodyc@ucdavis.edu
TITLE: Detection of Rare, Antigen-specific Human T cells with HLA Tetramers
AUTHORS: Zaki Molvi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>How I stain human T cells with tetramers following the guidelines from Sewell's group: </span><a href="https://pubmed.ncbi.nlm.nih.gov/26076649" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://pubmed.ncbi.nlm.nih.gov/26076649/</span></a></div></div>

[STEPS]
?. [Staining]
Harvest cells of interest (PBMC, purified T cells, BMMC) either from culture or from frozen stock
?. [Staining]
Wash cells in PBS and resuspend in 50nM Dasatinib in PBS. Choose a volume appropriate with your number of samples in the next step. You will need 50uL of sample per tetramer. Incubate 37C for 30-60m.
?. [Staining]
Add 1uL directly of PE-conjugated tetramer to 50uL cells (still in Dasatinib from step 3). Incubate 30m on ice. The number of cells required will depend on the frequency of your population of interest. It is unlikely that your cells will be too dense at 50uL unless you are staining >50M lymphocytes.
?. [Staining]
Wash samples twice with FACS buffer (we use PBS + 2mM EDTA + 0.5% BSA + 0.1% w/v sodium azide). For multiple samples, performing washes in v-bottom plates is more convenient than tubes. Perform all spin steps at 860g, 4C, 4min.
?. [Staining]
After last spin, resuspend cells in 50uL per sample of anti-PE (diluted 1:100 in FACS buffer; clone PE001). Incubate 30m on ice.
?. [Staining]
Wash twice with FACS. Resuspend in 50uL PE-conjugated Goat anti-Mouse Ig (diluted 1:100 in FACS buffer; assuming you used PE001, a mouse isotype antibody). Incubate 30m on ice.
?. [Staining]
Wash twice with FACS. Resuspend in 50uL of surface stain (e.g. CD3, CD8, CD4, as well as a dump channel including  CD16 CD19 CD56 CD14. Differentiation markers can be included as well such as CD62L, CD45RA/RO, CCR7). Incubate 30m on ice.
?. [Staining]
Wash twice with FACS. After last wash, if using 7AAD as live/dead, resuspend cells in 7AAD diluted 1:100 in FACS buffer. Otherwise resuspend in FACS buffer. Strain cells and acquire.
?. [Acquisition]
If looking for low frequency populations, collect as many events as possible.
?. [Acquisition]
Gate on live, singlet, dump channel-negative, aggregate-negative CD3+ cells before looking at your Tetramer+ve population. When looking for rare populations, do not be generous with singlet, live/dead, scatter, or phenotypic gates. It is crucial to exclude noisy outliers such as fluorophore aggregates. Exercise caution when choosing to include CD4+CD8+ over single positives.
TITLE: CRISPR knock-in validation
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
CRISPR knock-in validation

[STEPS]
1. 



Cultured hippocampal neurons tagged with oScarlet were prepared with Lucigen
QuickExtract DNA extraction solution (Biosearch Technologies, cat# QE09050).
Briefly, neurons were lysed by adding 50 ul of QuickExtract solution to each
sample. 

2. The samples were mixed by pipetting and incubated at 68 °C for 15 min
followed by 95 °C for 10 min in a thermocycler before being stored at -20 °C
for downstream analysis. 





3. Two different PCR reactions were performed to amplify
DNA products of ~500 bp corresponding to the 5’ and 3’ integration junctions.
PCR#1 used the α-syn forward primer: TGTGCTTTCTCTTCCCTCTCTG and the reverse oScarlet primer CCGTCCTCGAAGTTCATCAC, whereas PCR#2 used the α-syn forward primer:
ATAACACTTCGTGCAGCACC and the reverse oScarlet primer ACAGGATGTCCCAGGAGAAG. 
4. PCR products were extracted from the agarose gel using the Monarch DNA gel
extraction kit (New England Biolabs, cat# T1020S), and samples were submitted for
sequencing for analysis at MCLAB.
TITLE: The Summary of Treatment Protocol in motivational psychotherapy
AUTHORS: Hosein  Sahebdel, Mohammad  Tahan

[DESCRIPTION]
Reviewing the history of theories associated with psychotherapy establishes that motivational psychotherapy (MPT) has been centered on environmental mind, lies, selfishness, and rich mind, which are often rooted in previous theories but portrayed in an effectively different manner. MPT disapproves of most theories of psychotherapy, wherein psychoanalysts, exaggerating the past, place individuals in very deep holes and indoctrinate them with some disgusting lies, and in fact, instill truths in their own minds and suggest a difficult way to cope with them. The analytical method has been also exploited to reflect on the theory of MPT. The results of this study revealed that MPT, recruiting the concept of rich mind, opens a new path for clients to effortlessly remove many problems in order to reach perfection and nurture their soul.

[STEPS]
1. Rapport> 	Rapport, providing introductory acquaintance, having discussion about the purpose of the therapy, building trust.
2. Describing> 	Helping clients to describe themselves by emphasizing incompatible aspects of their personality, this description includes all the characteristics of clients, including cognitive, emotional, moral, and so on.
3. Lies> 	Explain the concept of lies and how lies are formed. Describing and Explaining the Nature of Lies. Finding the root of lies. Separation of ugly and beautiful lies. Pathology of lies.
4. Roles in the script of life>	 Describe the script of life and analyzing his/her role in the script of life. Identify mental illness as a role. Pathology of roles.
5. Four walls> 	Four wall identification. Explaining the concept of four walls and try to break the first wall, using techniques such as Encouragement, Body language, Change from outside to inside, Confrontation with fear. Breaking sick habits, practicing: “I can”, breaking ugly lies, changing appearance.
6. Rich mind> 	Explaining the concept of rich mind and helping clients having rich mind, Get rid of trivial matters. Trying to control anger and calm. Searching for bigger goals.
7. Self-mastery> 	Practice self-mastery, Gain the power to change, Overcoming Ugly Lies: “i cant”. practicing new roles. Practice mastering habits and behaviors.
8. Personal circle> 	Practice personal circle and get rid of the hell of others. Practice loving themself. Reduce traumatic mental conflicts about others. Get rid of jealousy, revenge and anger. Following personal goals.
9. Commitment>  	Celebrating success, having commitment to change.
TITLE: DNA Isolation (Gel Clean-up)
AUTHORS: NUS iGEM

[DESCRIPTION]
2023 NUS-Singapore iGEM team followed this protocol to isolate the DNA fragments from the agarose gel after the gel electrophoresis. 

[STEPS]
1. Prepare and label an Eppondoft tube.
2. Place the agarose gel onto the LED Transilluminator (blue light) to observe the DNA band(s).
 

3. Cut out the target DNA band from the agarose gel.
4. Put the gel piece into the Eppendorf tube.
5. Add 450 µL of   into the Eppendorf tube.

6. Heat the Eppendorf tube at 55 °C for 20 min in the Thermo-Shaker.

7. Add 150 µL of 100% isopropanol (IPA) into the Eppendorf tube and shake the tube to mix the solution well.

8. Transfer the whole solution into a QIAquick Spin Column (purple tube with a maximum volume of 750 µL). 

9. Centrifuge it for 13 rpm, 1 min.

10. Discard the flow-through and place the QIAquick column back into the same tube.
11. Add 700 µL of   into the QIAquick column.

17. Centrifuge the emptied QIAquick column at 13 rpm, 1 min to remove residual  .

18. Transfer the QIAquick column into the newly labelled Eppendorf tube.
19. Add 30 µL of DI water into the QIAquick column.

20. Centrifuge the tube at 13 rpm, 1 min, ensuring that the direction of the Eppendorf tube’s cap is the same as the direction of spinning to avoid breaking.

21. Discard the QIAquick column, the solution left in the Eppendorf tube contains the DNA fragment of interest.
22. Use the Nanodrop to measure and record the purity and concentration of the DNA fragment.
 

12. Centrifuge it for 13 rpm, 1 min.

13. Discard the flow-through and place the QIAquick column back into the same tube.
14. Add 700 µL of   again into the QIAquick column.

15. Centrifuge it for 13 rpm, 1 min.

16. Discard the flow-through and place the QIAquick column back into the same tube.
23. Keep the isolated DNA fragment in the Room temperature.
TITLE: Differentiation of dendritic cells from human monocytes
AUTHORS: Girija Goyal

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the steps conventionally used to differentiate dendritic cells from human monocytes isolated from fresh whole blood or leukopaks. While these DC mimic many of the functions of the dendritic cell lineage, they are not considered classical DC which are a different lineage and very rare. By day 4 CD1c+ cells are emerging with a reduction in CD14 positivity</div></div>

[STEPS]
?. [Plate monocytes]
Plate monocytes in a 24 well plate with 1 million monocytes/well in 1 ml of RPMI+400ng/ml GM-CSF and 250ng/ml IL4.
?. [First media change]
2 days later (maximum 3 days), assess if there are any non-adherent, small cells floating around. These might be neutrophils or lymphocytes. If the number of these cells is high, it can impact the differentiation of DC. To remove non-adherent cell, gently aspirate all the media in the well and add fresh cytokine containing media. If the non-adherent cells are very few in number, proceed to step 3.
?. [Subsequent media changes]
If culturing past day 5, only replace half of the media by gently aspirating from the top and then adding fresh cytokine containing media.
?. [Dendritic cell collection]
Collect dendritic cells on day 5 or later by pipetting multiple times around the well to remove cells and then collecting the suspended cells.
TITLE: Background and Rationale (Part 1 of Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-onset Type 1 Diabetes Mellitus)
AUTHORS: Stephen.Gitelman , Jeffrey A. Bluestone

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is Part 1 of  "Safety and Efficacy of Imatinib for Preserving Beta-Cell Function in New-Onset Type 1 Diabetes Mellitus".</div><div class = "text-block">This clinical study is supported by JDRF. The aim of the collection is to determine whether imatinib will slow the progression of the autoimmune destruction of ß cells and lead to the preservation of C-peptide secretion in T1DM and to assess Diabetes-related objectives and safety of Imatinib in participants with new-onset type 1 diabetes mellitus".</div></div>

[STEPS]
TITLE: Protocolo Reto F1017B
AUTHORS: Valentina Lugo Moreno

[DESCRIPTION]
Este archivo es con el propósito de explicar los pasos para llevar a cabo el experimento "motor lineal" del bloque F1017B, detallando los materiales, pasos para hacer el prototipo y como llevar a cabo el experimento.

[BEFORE_START]
Conseguir los materiales.

[STEPS]
SECTION: Prototipo
1. Medir la tabla y los imanes, encontrando una relación entre ambos para darles la distancia necesaria (misma distancia entre cada imán y la madera 92mm x 30mm x 14 mm), realizar un boceto en la madera 400mm x 90mm x 20mm para tener fijo el lugar de cada material. 
SECTION: Prototipo
3. Con la cegueta hacer ranuras en las maderas 92mm x 30mm x 14mm, tomando en cuenta la medida del ancho de las varas de Soldadura 0% Plata Cluxer y la del largo de los imanes.
SECTION: Prototipo
4. Con pegamento "No más clavos" pegar los imanes y las dos piezas de madera  92mm x 30mm x 14mm  en su espacio correspondiente del boceto, dejándolos secar con intervalos de 15 minutos; al secar, colocar las varas de soldadura 0% Plata Cluxer en su espacio correspondiente.
SECTION: Prototipo
12. 
SECTION: Prototipo
2. Cortar 10 cm de termopar de boiler para gas LP y enderezarla hasta obtener una estabilidad.
SECTION: Experimento
6. Colocar el termopar de boiler para gas LP en el prototipo, conectar los cables caimán a la fuente de carga y a las varas de soldadura 0% Plata Cluxer
SECTION: Experimento
7. Dejar correr el prototipo y grabarlo.
SECTION: Experimento
8. Repetir 2 veces más.
SECTION: Tracker
9. Medir en tracker la posición vs tiempo.
SECTION: Prototipo
5.
TITLE: Proflexia RX Male Enhancement - [SCAM Or LEGIT] Report!
AUTHORS: V  V

[DESCRIPTION]
Proflexia RX: Does It Work? Alarming Research Revealed! Job – (DONOTUSE) University of California Santa Barbara Police Department.

[STEPS]
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TITLE: Immediate Early Gene (IEG) mapping of spinal cord neurons activated by cystometry-induced micturition in rats [keast-002]
AUTHORS: Janet Keast, Peregrine Osborne, Nicole Wiedmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection describes the procedures required to visualize and characterize lumbosacral spinal neurons that are activated by cystometry of awake adult male and female Sprague-Dawley rats. This collection includes protocols for:</div><div class = "text-block">STAGE 1: Surgery to cannulate the bladder, followed by recovery then cystometry</div><div class = "text-block">STAGE 2: Intracardiac perfusion with fixative to preserve the spinal cord tissue</div><div class = "text-block">STAGE 3: Immunohistochemical labelling of spinal cord sections to visualise immediate early gene expression in specific spinal regions and neuronal populations</div><div class = "text-block">STAGE 4: Microscopy and image analysis to assess patterns of immediate early gene expression in different spinal cord regions</div></div>

[STEPS]