eamag/protocols-with-steps huggingface dataset:
TITLE: Cyanobacteria Total Lipid Extraction from Polycarbonate Filters
AUTHORS: Robbie Martin, Steven W Wilhelm, Katarina A. Jones, Hector Castro, Shawn Campagna

[DESCRIPTION]
This protocol is designed/used for extraction of total cellular lipids from cyanobacteria samples (either lab cultures or field samples) collected on polycarbonate filters for use in lipid analysis and quantification via mass spectrometry. 



Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) or Robbie M. Martin (rmarti49@vols.utk.edu) for additional information regarding this protocol.
 
Modified from Guan, X. L., Riezman, I., Wenk, M. R., & Riezman, H. (2010).  Yeast lipid analysis and quantification by mass spectrometry.  Methods in Enzymology, 470, 369-391.
 

[STEPS]
20. Prepare the three separate solutions needed for this extraction protocol as listed below.
 lipid extraction solvent (step 2) with a 1:1 ratio of butanol and lab purified water (water-saturated butanol, and lab purified water
 
1. Prepare the three separate solutions needed for this extraction protocol as follows:
lipid extraction solvent: a 15 : 15 : 5 : 1 : 0.18 ratio by volume of 95% ethanol, water, diethyl ether, pyridine, and 4.2 N ammonium hydroxide, respectively.
water-saturated butanol:  a 1:1 ratio of butanol and Milli-Q water
purified lab water: (Milli-Q water) 
2. Unfold polycarbonate filter and place into a 2-mL centrifuge tube with cell side of filter facing outwards.

Note: Appropriate volume of lab culture or field samples to filter and extract depends on cell concentration.  As a guideline, we have been successful filtering 10-25 mL of lab cultures of Microcystis aeruginosa and ~50 mL of either raw lake water or mesocosm samples.
3. Add 1 mL of extraction solvent, ~100 µL of glass beads, and vortex ~5 s. 
4. Incubate sample in 60 °C water bath for 20 min.
5. Centrifuge sample at 10,000 x g for 10 min.
6. Remove supernatant and place into a 1-dram glass vial (dram vial #1).  The first two extractions from a sample will be placed in this vial (#1).
7. Repeat steps 3-6, except DO NOT ADD more glass beads.
8. Dry the collected supernatant in dram vial #1 under a stream of nitrogen.
9. Re-suspend dried sample in 300 µL of water-saturated butanol and 150 µL of Milli-Q water.
10. Vortex and transfer to a 2-mL centrifuge tube.
11. Centrifuge at 10,000 x g for 2 min. 
12. Remove top butanol phase and place into a NEW 1-dram glass vial (dram vial #2).
13. Wash original dram vial (#1) with 300 µL saturated butanol and transfer to residual aqueous phase in 2-mL centrifuge tube from step 10.  Vortex.
14. Centrifuge at 10,000 x g for 2 min.  Remove top butanol phase and place into dram vial #2.
15. Dry the collected butanol phase in dram vial #2 under a stream of nitrogen.
16. Re-suspend dried sample in 300 µL of 9:1 methanol:chloroform.
17. The samples are now ready for analysis via LC/MS.
TITLE: Actin flow 
AUTHORS: Juan.Gonzalez

[DESCRIPTION]
Actin flow

[STEPS]
SECTION: Experimental procedure
1. Transfect the cells with the LifeAct-GFP plasmid following the protocol "Cells electroporation for cell transfection with NEON system"


SECTION: Experimental procedure
2. The next day, trypsinize the cells as usual

SECTION: Experimental procedure
3. Seed the cells on glass (i.e. gels on glass bottom petri dishes or the glass itself)


 

SECTION: Experimental procedure
4. After 180 min , image the cells with a confocal microscope. Use the 40x objective. If the CO2 cannot be controlled, use CO2-independent medium. The channel would be eGFP. 

SECTION: Experimental procedure
5. Focus the cell in a focal plane where the actin cytoskeleton can be seen


SECTION: Experimental procedure
6. We perform a time series of 120 images every 2 s (4 min in total per cell)
SECTION: Data analysis
7. 
Open the image sequence with Fiji software



SECTION: Data analysis
9. 
Analyze, Set measurements, select “Bounding rectangle” and “Min & Max gray value”



SECTION: Data analysis
10. 
Check where the cell cytoskeleton moves



SECTION: Data analysis
11. 
Draw a line with the wand tool crossing the movement



SECTION: Data analysis
12. 
Analyze, Multi kimograph, multi kymograph, linewidth: 1



SECTION: Data analysis
13. 
A black & white gradient pops up. White lines indicate cell movement. The higher angle indicates more movement



SECTION: Data analysis
14. 
Draw a line with the wand tool in the white line seen in the kymograph






SECTION: Data analysis
16. Press “M” and copy & paste the measurements in an Excel file 



SECTION: Data analysis
17. In the Excel file, split the width by the length. This new value means flow in "pixels per frame"
SECTION: Data analysis
18. Divide the pixels/frame value obtained by 2. This new value means flow in "pixels per second"
SECTION: Data analysis
19. Split the "pixels/s" value by the "pixels/micron" ratio of the objective used (i.e. 3.2055 in the LSM900 confocal microscope) and multiply by 1000. This final value is the actin flow in nm/s
SECTION: Data analysis
15. 

 


SECTION: Data analysis
8.
TITLE:       ELISA for quantification of IL-38 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>Il-38 acts on T cells and inhibits the synthesis of IL-17 and IL-22.  The placenta, tonsil's B lymphocytes, spleen, skin, and thymus widely express IL-38. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-38 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum. Human IL-38 present in the serum sample binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-38 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-38 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-38 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-38 standard dilutions and the human IL-38 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: New Bung Decontamination
AUTHORS: jack.hocking

[DESCRIPTION]
This protocol is the process by which we eliminate any stubborn contaminations picked up in the manufacture and/or shipping of our OGI Bio BioReactor Culture and Media Flask Bungs to our site.  

[BEFORE_START]
Ensure adequate PPE.

[GUIDELINES]
Virkon should be handled with care, following the PPE recommendations in the COSHH form.

[STEPS]
SECTION: On Receipt of Bungs
1. Place bungs in a large plastic container.
SECTION: On Receipt of Bungs
2. Fill container with tap water by decanting 1 or 2 L bottles into it.  Make a note of how many litres it takes to cover the bungs with at least a centimeter of water.
SECTION: On Receipt of Bungs
3. Per litre of water, add two virkon tablets to the container and wait for them to dissolve.
SECTION: On Receipt of Bungs
4. Once dissolved, agitate the liquid gently to ensure the virkon is well distributed.
SECTION: On Receipt of Bungs
5. Leave bungs to soak in virkon for 48 hours.
SECTION: Rinsing and Storage
6. Dilute virkon may be disposed of down a lab sink with tap running.
SECTION: Rinsing and Storage
7. Bungs should be rinsed with several loads of water, agitate the liquid gently to ensure needles are cleaned.
SECTION: Rinsing and Storage
8. Store bungs in a separate container uncovered.  Allow container to air-dry and store safely.
TITLE: Banana sensor sfGFP test
AUTHORS: Hung Liang Pai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The goal of this protocol is to ensure that our lab technique and environmental circumstarnces are compatible to this expiriment. Based on iGEM team EPFL 2019, we try to define a concentration of Toehold switch and trigger DNA sequence which has a high protein expression(sfGFP in this case) but low or no leakage at same time.</div></div>

[STEPS]
?. [Preparation]
Sterilize the bench, and put on a labmat
?. [Preparation]
Thaw the reagents
on ice
?. [Preparation]
Set the microplate (384 well plate) reader machine
?. [Protocols]
Spin down or gently shake the tube before adding (especially solution A and DNA plasmid)
?. [Protocols]
Separate the droplets in the different corners of the well (as the picture below)
?. [Protocols]
Seal the plate with microseal
on ice
?. [Protocols]
Centrifuge ,,
Centrifuge: 4000 33
4 °C
?. [Protocols]
Place the plate in the readers, make sure the setting is correct, and start the measurement
?. [Protocols]
After  of measurement, save the data, and turn off all the machines
TITLE: A multi-modal cognitive behavioral therapy training program for caregivers of children with disabilities in Uganda: a study protocol for a cluster-randomized controlled trial
AUTHORS: Mariam Namasaba, Ali Ayub Baguwemu, Sumaya Nabunje

[DESCRIPTION]
Background: Caregivers of children with disabilities (CWDs) often feel inadequate in providing proper care for their children. Some caregivers in schools report negative feelings toward CWDs and poor mental well-being. In the home, caregivers may fail to recognize their children’s unique needs, elevating their stress. Although cognitive behavioral therapy training programs have a moderate effect on improving mental well-being and building a sense of self-efficacy, no program has attempted to combine both the protective and ecological factors for improving caregivers’ mental well-being. 
Aims: This study aims to evaluate the effect of a multi-modal cognitive behavioral therapy training program on mental well-being, coping skills, and sense of self-efficacy among caregivers of CWDs in Uganda.
Methods: The study will be a cluster-randomized, controlled trial where twelve schools of CWDs will be randomly allocated to either the intervention or control arm. A sample of 400 home caregivers and 80 school caregivers will be assigned to either arm. A six-month multi-modal cognitive behavioral therapy training program will be conducted as an intervention, and caregivers in the control arm will receive treatment as usual. The study’s primary outcome will be caregivers’ mental well-being, and the secondary outcomes will be coping skills and a sense of self-efficacy. 
Conclusion: This study will guide policymakers in designing effective interventions that benefit the health of CWDs and their caregivers. The study results will further provide insight into the gaps in care provided to CWDs in school and home settings in Uganda.
Trial registration: UMIN-CTR Clinical trial (https://upload.umin.ac.jp/cgi-bin/ctr_e/ctr_view_reg.cgi?recptno=R000046712. 



[STEPS]
1.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>To setup the docking, the Lamarckian genetic algorithm (LGA) was used, and docked conformations were compared and clustered based on the conformational similarities and root-mean-square positional deviation (rmsd).  The numbers of runs were adjusted to 30 for all ligands. Rmsd was calculated based on rmsd conformational clustering tolerance of 2.0 Å. For genetic algorithms, parameters were setup as 150, 0.02, 0.8, 27000, and 2500000 for population size, mutation rate, crossover rate, maximum number of generation, and maximum number of energy evaluations, respectively. For LGA docking, the pseudo-Solis and Wets local search method was used with a maximum of 300 iterations per local search, and the probability of performing local search on an individual in the population was adjusted to 0.06, the size of local search space to sample, ρ, was 1.0, the maximum number of consecutive successes or failures before doubling or halving the ρ was four for both, and finally the lower bound on ρ was 0.01. </p>
<p> </p>
<p>To have access to sample data and configuration files also check out: doi: <a href="https://doi.org/10.13140/RG.2.2.20407.42403" target="_blank">10.13140/RG.2.2.20407.42403</a></p>

[BEFORE_START]
<p>Install all required software:</p>
<p> </p>
<p>MGL tools 4.2</p>
<p>UCSF Chimera</p>
<p> Accelrys Discovery Studio</p>
<p>Open babel</p>
<p>Cygwin</p>
<p>ChemSketch</p>
<p>SPDV Swiss PDBVIEWER</p>

[GUIDELINES]
<p style="text-align: justify;">For each target, you need a separate folder in which you have folders of compounds (ligands). For each ligand, separate docking algorithm should be run.</p>

[STEPS]
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TITLE: Sex Work and Sexual Violence Research Study Protocol 2022
AUTHORS: Scoular, Jane, Sanders, Teela, Abel, Gillian, Brents, Barbara, Ellison, Graham, Susie Balderston, Marriott, Nigel

[DESCRIPTION]
Globally, the most important public health issue that sex workers face is their experience of high levels of violence (Kinnell 2006, 2008; Alexander, 1999) with a systematic review estimating levels of sexual violence as being between 15 and 55% (Deering et al, 2014). 

The overarching question this study seeks to address is how social, legal, and judicial contexts shape the safety and well-being of people engaging in sex work and, in particular, how context shapes experiences of sexual violence.  This mixed method research will compare four different legal environments: legalisation (Nevada USA), criminalisation (Northern Ireland), decriminalisation (New Zealand) and partial criminalisation (England and Scotland). 

Interviews, a survey of sex workers and reviews of legal cases in each jurisdiction will generate empirical data from which to build theory as to:
- The relationship between legal consciousness, legal norms, and legal practices and experiences of sexual violence and sexual autonomy in different models of governance;
- How marginalised groups (sex workers) interpret the sexual violence they experience, their rights regarding sexual violence, how and whether they seek redress through the criminal justice system, and outcomes in different models of governance;
- How various criminal justice institutions respond to sexual violence among various sex-working populations in both theory (law and legal norms re consent) and practice (police actions, reporting systems, court dispensation) in different models of governance;
- Differences among groups of sex workers by gender, age, ethnicity, sexual orientation, citizenship, and sex market, especially for trans, MSM sex workers, and migrant communities;
- How researchers and expert advisors can work together in the research process to interpret findings and build theory, and empower expert advisors with evidence for better practices;
- Generate and disseminate evidence-based information on best practices to help provide justice for sex workers. 

The research will have ethical approval from each research site, an Advisory Board and trained, paid peer researchers to assist with data gathering, analysis and dissemination.  The study will report findings in 2023/2024.  It is funded by the Economic and Social Research Council. 







[STEPS]
TITLE: Usage of EMBL2checklists
AUTHORS: Michael Gruenstaeudl

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The submission of DNA sequences to public sequence databases is an essential, but insufficiently automated step in the process of generating and disseminating novel DNA sequence data.  A user-friendly software tool is needed that streamlines the file preparation for database submissions of DNA sequences that are commonly generated in plant and fungal DNA barcoding.  A Python package was developed that converts DNA sequences from the common EMBL and GenBank flat file formats to submission-ready, tab-delimited spreadsheets (so-called "checklists") for a subsequent upload to the annotated sequence section of the European Nucleotide Archive (ENA). The package, titled "EMBL2checklists", automatically converts DNA sequences, their annotation features, and associated metadata into the idiosyncratic format of marker-specific ENA checklists and, thus, generates output that can be uploaded via the interactive Webin submission system of ENA. Here, we present a step-by-step protocol of the bioinformatic steps necessary to generate submission-ready checklist files from raw DNA sequence data and associated metadata via EMBL2checklists.</div></div>

[STEPS]
?. Annotation of DNA sequencesAdd sequence features and feature qualifiers to each DNA sequence using INSDC-compatible feature table keywords.Use any of the following software tools:
?. Saving sequences as flat fileSave multiple DNA sequences of the same barcoding marker as an single, multi-sequence flat file in EMBL or GenBank format. Use any of the following software tools:
?. Validation of flat fileTest the validity of the file format, the feature table syntax or the taxonomic status of organism names, among other aspects, using one of two software tools.For validation of EMBL-formatted flat files, use:For validation of GenBank-formatted flat files, use:
?. Installation of EMBL2checklistsIt is recommended that you install EMBL2checklists via pip (i.e., the recommended installer of the Python Package Index). On Linux and MacOS, this can be achieved via the following command:Conversion from flat file to checklistConvert the EMBL- or GenBank-formatted flat file into the Webin checklist file format using the software:
?. Upload checklist to ENAUpload the resulting checklist to the sequence section of ENA using the interactive route of the Webin submission system.
TITLE: Rodent Gait Analysis Using Linear Track Protocol
AUTHORS: Rose B. Creed, Alexandra Nelson

[DESCRIPTION]
This protocol is used to evaluate quantitative aspects of mouse locomotor behavior (gait).

 


[STEPS]
SECTION: Procedure
1. There is no need to habituate mice to the apparatus; mice will walk across voluntarily to enter the black box.
SECTION: Procedure
2. Start video recording.
SECTION: Procedure
3. Place mouse on open end of the catwalk; observe to make sure mouse walks towards the black “home” box.
SECTION: Procedure
4. When mouse reaches “home” box, stop video recording. 
SECTION: Procedure
5. Remove mouse from “home” box and re-place on the open end. 
SECTION: Procedure
6. Repeat this process 4-5 times (or until mouse stops traversing catwalk). A greater number of trials yields a higher number of strides for subsequent analysis.
SECTION: Procedure
7. After final trial, return mouse to home cage. 
SECTION: Procedure
8. Data can be analyzed by following the steps described in: https://github.com/UCSF-Nelson-Lab/Catwalk-Analysi
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A real-time PCR to detect RNA in mouse liver tissue. This method has been adapted from a<br />publication by Liu XJ et al 2014, and the oligonucleotides have been modified and a different PCR kit<br />used.</p>

[BEFORE_START]
<p>If using a different brand or model of real-time thermocycler, check the concentration of ROX is<br />adequate.<br />Method assumes the user is familiar with the thermocycler and software used to run the<br />protocol and with PCR in general.</p>

[STEPS]
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TITLE: Lymphocyte proliferation in poultry species
AUTHORS: Ahmed O.Abass, Gamal Mehaisen

[STEPS]
?. The heparinized blood samples were added to separation medium Histopaque®-1077 (cat# 10771, Sigma, USA).
?. Samples were centrifuged at 1030 xg for 20 min at 4°C.
?. Peripheral blood mononuclear cells (PBMCs) were isolated and washed twice with RPMI-1640 (Invitrogen Corp., Grand Island, NY, USA) and then re-suspended in 2 ml of RPMI-1640 complete culture medium.
?. The viable lymphocytes were detected using Trypan Blue dye and plated in triplicate wells (96-well plate) at 1×106 cells per well.
?. A 50 μl of either Concanavalin-A (Con-A, 45 μg/ml, cat# C5275, Sigma, USA) or Lipopolysaccharide (LPS, 10 μg/ml, cat# L4391, Sigma, USA) was added to selected wells to induce the proliferation of T lymphocyte and B lymphocyte, respectively.
?. Control wells received 50 μl of RPMI-1640 medium.
?. Cells were then incubated for 48 h at 42 °C with 5 % CO2.
?. After incubation, 15 μl of 3-[4,5-dimethylthiazol]-2,5-diphenyltetrazolium bromide (MTT, 5 mg/ml, cat# M2128 , Sigma, USA) was added to each well and the cells were incubated for another 4 h.
?. Subsequently, 100 μl of 10% sodium dodecyl sulfate dissolved in 0.04 M HCl solution was added to each well to lyse the cells and solubilize the MTT crystals.
?. Finally, the absorbance at 570 nm was recorded using an automated ELISA microplate reader (ChroMate® Microplate Reader-4300, Awareness Technology Inc., Palm City, FL, USA).
?. Stimulating index (SI) for either T or B cells was calculated as follows: SI = OD570 (stimulated cells) / OD570 (unstimulated cells).