eamag/protocols-with-steps huggingface dataset:
TITLE: RNA extraction protocol for snake genomes using TRlZOL reagent（Invitrogen）
AUTHORS: Boyang Liu, Liangyu Cui, Zhangwen Deng, Yue Ma, Diancheng Yang, Yanan Gong, Yanchun Xu, Shuhui Yang, Song Huang

[DESCRIPTION]
A protocol to extract RNA from snake muscle using TRlzol reagent (Invitrogen, USA) and RNA extraction kit (Thermo Scientific, USA) in this protocol.

[STEPS]
SECTION: RNA extraction
1. The laboratory and test bench for RNA extraction need to be sterilized using alcohol and ultraviolet light for 30 minutes. 
30 min 

All the equipment used in the experiment, such as mortar and pestle, are sterilized at high temperature one day before the experiment and dried in an oven. 

Masks and gloves need to be worn throughout the RNA extraction process. Avoid exposure of RNases to experimental samples and cause RNA degradation.
SECTION: RNA extraction
2. On the clean bench, take 50 mg of scented glands and put them into a pre-cooled mortar to ensure that the liquid nitrogen has never passed the sample, quickly grind the sample into powder, add 1 ml TRIZOL, mix the tissue powder with TRIZOL, and put it into In a centrifuge tube, stand at room temperature for 15 min.
Room temperature 15 min 

SECTION: RNA extraction
3. Add 200 ml of chloroform, shake the centrifuge tube for 15 s to mix the chloroform and TRIZOL well.
Stand at room temperature for 10 min, put it in a refrigerated centrifuge and centrifuge at 4°C, 12000r/min, 15 min.
15 s 
Room temperature 10 min 
   

SECTION: RNA extraction
4. After centrifugation, the solution should be separated into three phases. Take the upper aqueous phase into a new centrifuge tube, add three times the volume of ethanol to mix, and then stand at room temperature for 10 min.
Room temperature 10 min 

SECTION: RNA extraction
5. Add the mixed solution to the purification column, centrifuge at 12000r/min at 4°C for 2 min, repeat three times to ensure that ethanol is removed.
  

SECTION: RNA extraction
6. RNA is purified with an RNA extraction kit (Thermo Scientific, USA), and RNA was eluted with DEPC.
SECTION: RNA extraction
7. Add RNase inhibitors to ensure that the RNA will not be degraded, and mark the outside of the centrifuge tube.

Check RNA purity and concentration, and confirm RNA integrity using electrophoresis.
TITLE: Yale - Blood or Urine Calcium
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to determine the concentration of calcium in blood, serum, and plasma. Calcium is measured as the complex with arsenazo III and monitored at 600nm.</div></div>

[STEPS]
?. Calibrate Cobas for Calcium analysis by running a multi analyte standard and two assayed control serum.
?. Sample handling as performed by the Cobas Mira Plus.    a) Pipette 5µL of sample into a cuvette slot.    b) Add 180 µL of Calcium Liquid Reagent.    c) Mixture is incubated at 37˚C and spun for 10 minutes.    d) Absorbance is measured at 650nm.
TITLE: MDL-LDA Agile Method
AUTHORS: Freddie Prianes

[DESCRIPTION]
As a guide in modeling the document labels using Latent Dirichlet allocation for archived documents in an Integrated Quality Assurance System (IQAS), the researchers used the agile method as it promotes flexibility, speed, and, most importantly, continuous improvement in developing, testing, documenting, and even after delivery of the software. Since the phases of this model are light, the teams are not bound by a rigid systematic-based process on pre-set constraints and restrictions as some other models, like the waterfall model, and can adjust changes whenever they are needed. This flexibility on every stage propagates creativity and freedom within processes. Furthermore, development teams can modify and re-prioritize the backlog, allowing for speedy implementation

[STEPS]
SECTION: MDL-LDA Methodology
1. Plan: the researchers collected previous documents involved in the accreditation process, such as compliance reports under the areas of student, faculty, facility, library, and administration. Also, understanding of the existing problems in tracking, tagging, and duplication of these documents during the accreditation process.
SECTION: MDL-LDA Methodology
2. Design: the requirement specifications in this stage were identified in relation to the existing problem of the HEI in tracking, tagging, and duplication of the documents for accreditation and quality assurance. Along with this, the researchers also created the process of the intelligent model, which will be the basis of document labelling.
SECTION: MDL-LDA Methodology
3. Develop: this stage is intended on the creation of the prototype, which involves the processing of the documents in order to identify the proper label for each document.
SECTION: MDL-LDA Methodology
4. Deploy: the prototype undergoes a test run during this stage.
SECTION: MDL-LDA Methodology
5. Review: the researchers conduct a checklist function review to check if each component is running properly.
SECTION: MDL-LDA Methodology
6. Launch: wherein the prototype is embedded to the local system of the HEI.
TITLE: Title Abstract Screening in Sysrev
AUTHORS: Melanie Gainey

[DESCRIPTION]
This protocol contains instructions on how to do the first stage of screening, called title abstract screening, in the Sysrev platform. Contact Melanie Gainey (mgainey@andrew.cmu.edu) if you are having any issues or have questions.


[STEPS]
1. Create an account in Sysrev (https://sysrev.com/).
2. Use this link to join our project: [insert link to project]
3. These instructions and the Inclusion Exclusion criteria are linked in the Project Description field for easy access.

 
4. Click on Review on the home screen to begin screening articles.

 
5. Read the title and abstract of the article the platform is showing you and make a Yes or No decision to include based on the inclusion and exclusion criteria and hit the Save button. Sysrev will feed you the next article.

 
6. Some things to keep in mind as you screen:
If you are unsure whether an article should be included or not, select Yes. An inaccurate Yes decision can be fixed later, while an inaccurate No decision is not fixable as those articles are permanently removed from the project.



6.1. Monitoring your progress:
It should take on average 3 minutes to screen each article - this is a rough estimate.
You can check how many articles you’ve screened on the Member Activity panel on the home screen (Overview tab).

6.3. Blind Review:
A key element of evidence synthesis projects is to reduce bias by using two independent and blind reviewers to make decisions (meaning that they are not aware of the other reviewer's decision). If you click on the Articles tab, you will be able to see other people's decisions. We want to make independent blinded decisions, so avoid looking at that tab as you screen.
6.2. Incomplete, Non-English, or Duplicate Records:
If an article does not have an abstract, you can try to find the article on google and copy and paste it into the Notes field. If you are still unable to find it, select Yes.
If the article is not in English, you can try putting it in Google translate (copy and paste the English version in the Notes field). If still not sure, select Yes.
If you notice duplicate titles, you can select Yes or No based on the inclusion criteria and then add a note in the Notes field that it is a duplicate.
TITLE: Abundance  of  fungal  hyphae  in  seawater  by  epifluorescence  microscopy  using  Calcofluor  White  stain  method
AUTHORS: M.H.  Gutiérrez

[DESCRIPTION]
Protocol to stain fungal hyphae from seawater with Calcofluor White.

[GUIDELINES]
References
 
Damare S, Raghukumar C (2008) Fungi and macroaggregation in deep-sea sediments. Microb Ecol 56:168–177
 
Jebarag CS, Raghukumar C (2009) Anaerobic denitrification in fungi from the coastal marine sediments off Goa, India. Mycol Res 113:100-109
 
Newell, S.Y., Statzell-Tallman, A. (1982) Factors for conversion of fungal biovolume values to biomass, carbon and nitrogen: variation with mycelial ages, growth conditions, and strains of fungi from a salt marsh. Oikos 39: 261-268
 
Rasconi S, Jobard M, Jouve L, Sime-Ngando T (2009) Use of calcofluor white for detection, identification, and quantification of phytoplanktonic fungal parasites. Appl Environ Microbiol 75:2545-2553

[STEPS]
SECTION: Collection
1. Collect samples in sterile 50 mL polypropylene tubes and fix with formaldehyde or glutaraldehyde (2% final concentration). Store samples at 4 °C in the dark.

SECTION: Stain
3. Stain filters with the retained material from filtration by applying 600 µLof aqueous 0.1% Calcofluor White directly to sample and filter, making sure to cover the entire area of the filter.

Allow 5 minto 10 min then use vacuum to remove the excess stain from the filter. Avoid any light source.



SECTION: Filtration
2. Filter 5 mL to 30 mL (depending on the environment) of seawater by 0.22-µm mesh black 25 mm diameter polycarbonate filters (Millipore Corp.). 


SECTION: Abundance
4. Place the filter (sample side up) onto a slide and add 1 drop of non-fluorescent immersion oil to the top of the filter, then cover with a cover slip.

SECTION: Abundance
5.  Count slides immediately on epifluorescent microscope or store at -20 °C.


SECTION: Abundance
6. Use an epifluorescence microscope equipped with UV filter (used for DAPI, eg. filter set 49 Carl Zeiss Ltd., 365 nm excitation and 445-450 nm emission band pass) to examine at 1000X the entire effective area of the filters. 
SECTION: Abundance
7. Count all hyphae identified and record their length and width. Use cylinder volume as a morphological approximation to estimate the biovolume of fungal filaments.
TITLE: untitled protocol
AUTHORS: Khairil Asnan, Mohd. Sadzan Asram bin Aliming, Aprialdy Idrus, Diana Eka Pratiwi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span>Cattle's blood component is compressed using centrifuge (5000 rpm for 10 minutes), then supernatant (erythrocytes) collected. Erythrocytes with a ratio of 3:1 (serum:supernatant), treated with initial mixing each with HCl (0.5 M) NaOH (0.5 M), and 30% H</span><span style = "vertical-align:sub;">2</span><span style = "vertical-align:sub;vertical-align:sub;">O</span><span style = "vertical-align:sub;">2</span><span>with an initial ratio of 1:1 (v/v). Each variable then treated by mixing SnCl</span><span style = "vertical-align:sub;">2</span><span>(1 M, 1:1 v/v), oleic acid (2:1 v/v), and O</span><span style = "vertical-align:sub;">2(g)</span><span> (0.5 L/m, 60s). The best sample then purified by heat about 800</span><span style = "vertical-align:super;">o</span><span>C for 120 minutes.The highest calculated total iron mass is 240000 μg/100 ml (NaOH + oleic acid). Sample purification has increased the iron concentration up to 46.30% (m/m%).</span></div><style>
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[STEPS]
?. [Sample preparation]
Compress the whole blood sample from cattle.
6 ml
37 °C
?. [Sample preparation]
Mix  sample with
[blood sample]
[NaOH]
37 °C
dark green solution with strong odor
?. Chelate reaction by adding  into the previous mixed sample solution then let it sit for . After that, mixed them by  for  .
[Oleic acid]
TITLE:   ELISA for quantification of IL-5 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>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. They have both paracrine and autocrine function. Interleukins are also used in animal studies to investigate aspect related to clinical medicine.T cells and stem cells make IL-3. It functions as a multilineage colony-stimulating factor. CD4+T cells (Th2) produce IL-5, and its principal targets are B cells. It causes B-cell growth factor and differentiation and IgA selection. Besides, causes eosinophil activation and increased production of these innate immune cells </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-5 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl  of human serum. Human IL-5 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash o remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-5 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 binds to the biotin-conjugated anti-human IL-5 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) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-5 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-5 standard dilutions and the human IL-5 sample concentration determined.
?. For better results place the microplate on a plate shaker in every incubation.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Genomic DNA was extracted from white blood cells (PAXgene-RNA tubes) using the PAXgene blood RNA kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions (PAXgene blood RNA kit handbook version 2, April 2008). Taget gene and adjacent DNA exons and SNP analyses were conducted using PRISM3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA) and TaqMan SNP Genotyping Assay (Applied Biosystems) according to the manufacturer's instructions. (Hokkaido System Science, Sapporo, Japan).</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A basic Western Blot protocol that has been optimized specifically for cell lysates.</p>

[BEFORE_START]
<p>You will need to optimize the weight of your running gel before you begin. Most proteins will be separated with 8%-12% acrylamide gels. If your protein runs off your gel, you will need to use a heavier gel. See step 12 for more information.</p>

[STEPS]
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TITLE: Human_Tissue_Nuclei_Isolation_Protocol_2021_10_18
AUTHORS: Kimberly Paquette

[DESCRIPTION]
Homebrew protocol to isolate nuclei from human frozen brain tissue.

[STEPS]
SECTION: Working Solutions: PBSTA (3mL) without TritonX-100
3. 
 

 

      Solution  Final Conc  1 RXN  5.2 RXN    10x PBS  1x  300uL  1560uL    1M MgCl2  3mM  9uL  46.8uL    D-Sucrose (342.29)  0.3M  0.3081g  1.602g    ** RNasein (add on day)  0.4 U/uL  30uL  156uL    Ultrapure   Water  Fill to: 3mL  Fill to: 15.6mL  
 Add most of the Ultrapure water, then PBS, then MgCl2, then Sucrose.
SECTION: Working Solutions: PBSTA (3mL) without TritonX-100
4. Vortex to dissolve.
SECTION: Working Solutions: PBSTA (3mL) without TritonX-100
5. Then add Ultrapure water to final volume.
SECTION: Working Solutions: PBSTA (3mL) without TritonX-100
6. Add RNasein before using.
SECTION: Working Solutions: 1.4 M PBS Cushion (8 mL)
7.  
 

      Solution  Final Conc  1 RXN  5.2 RXN    10x PBS  1x  0.8 mL  4.16 mL    1M MgCl2  3 mM  24 uL  124.8 uL    D-Sucrose  1.4 M  3.8336 g  19.935g    10% Triton X-100  0.10%  80 uL  416 uL    Ultrapure   Water  Fill to: 8 mL  Fill to: 41.6 mL  
 Add half of the Ultrapure water, then PBS, then MgCl2, then Sucrose.
SECTION: Working Solutions: 1.4 M PBS Cushion (8 mL)
8. Vortex to dissolve.
SECTION: Working Solutions: 1.4 M PBS Cushion (8 mL)
9. Then add 10% Triton.
SECTION: Working Solutions: 1.4 M PBS Cushion (8 mL)
10. Then add Ultrapure water to final volume.
SECTION: Working Solutions: Nuclei Wash and Resuspension Buffer
11. 
 
 
      Solution  Final Conc  1 RXN  5.2 RXN    10x PBS  1x  400 uL  2080 uL    50mg/uL Ultrapure BSA  1%  800uL (40mg)  4160 uL    ** RNasein (add on day)  0.2 U/uL  20 uL  104 uL    Ultrapure   Water  Fill to: 4 mL  Fill to: 20.8 mL  
 Add most of the Ultrapure water, then PBS, then BSA.
SECTION: Working Solutions: Nuclei Wash and Resuspension Buffer
12. Tilt to dissolve, do not vortex.
SECTION: Working Solutions: Nuclei Wash and Resuspension Buffer
13. Add RNasein before using.
SECTION: Stock Solutions
1. 10% Triton X-100 (100 mL) 
      Solution  Final Conc  Volume  Notes    Triton X-100  10%  10mL      Ultrapure   Water  Fill to: 100mL  
 

SECTION: Stock Solutions
2. 1 Molarity (M) MgCl2 (100 mL) 
      Solution  Final Conc  Volume  Notes    MgCl2 (FW 203.31)  1M  20.331g  203.31g/1000mL = 1M 5.08275g/100mL = 1M    Ultrapure   Water  Fill to: 100mL
TITLE: Safety Study of Wireless Fecobionics Device
AUTHORS: Yanmin Wang, Ghassan Kassab, Hans Gregersen, Patel Bhavesh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We developed a novel wireless device (Fecobionics) for mapping colonic and anorectal neuromuscular function. Our hypothesis is that the Fecobionics device will be safe to use for repeated testing without caused mucosal damage or any unwanted adverse effects. Validation of this innovative approach map and describe objectively (without disturbing the colonic transport and defecation processes) the transport characteristics and neuromuscular signatures during colonic transport and initial entry from the rectum into the relaxing anal canal.</div><div class = "text-block">Twelve Mongrel dogs will be assigned into two groups: Fecobionics group and predicate control group (N=6 of each). Fecobionics and the predicate device will be transanally inserted into rectum. Further, each group is divided into two sub-groups: The acute group in which the animals will be euthanized immediately after the evacuation of the device, and the chronic group in which the animals will be terminated after 2 weeks. The animals will be monitored daily. After termination, colon and rectum will be harvested for histological measurements.</div></div>

[STEPS]
TITLE: Measuring non-healthcare occupational exposure to SARS-CoV-2 across occupational groups in the United States
AUTHORS: Michael Walsh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol explains how to characterize non-healthcare occupational exposure to SARS-CoV-2 across occupational groups in the United States using the SARS-CoV-2 Occupational Exposure Matrix (SOEM).</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div></div>

[STEPS]
?. Implement the steps outlined in Protocol_Final_Walsh_2021.pdf. (Attached)
TITLE: Colony formation assay in Matrigel
AUTHORS: Goran  Tomic

[DESCRIPTION]
This is a colony formation assay that I have set up as an alternative to soft-agar assay. It is based on a 3D on-top assay (Lee et al. 2007, 4, 4, 359) https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933182/

[STEPS]
1. 
Thaw Matrigel on ice at 4 C overnight. Prechill one 48-well plate, 200 uL box of tips.

2. Coat prechilled culture surface with a thin layer of Matrigel (80 uL per well in a 48-well plate)
Incubate for 20 min at 37C to allow to gel
3. Trypsinise cells (Filter through a 40 um mesh to make single cells)
Aliquot cells in a 1.5 mL eppendorf tube to have 2500 cells/100 uL, multiplied by the number of replicates, and spin down at 1200 rpm. Depending on the cell line, optimisation of cell number might be needed. 
4. Add 100 uL of cell suspension on top of Matrigel in wells. 
Leave for 20 min at 37C for cells to attach to Matrigel. 
5. Chill the remaining medium on ice and add Matrigel to 10%
Gently add 100 uL of the 10% Matrigel medium on top
6. Maintain culture for 4 days. Change the medium every 2 days (with 10% Matrigel)
7. Observe the formation of colonies. Once ready for imaging, scan on Oxford Optronics colony counter or another method of choice. https://www.oxford-optronix.com/gelcount-cell-colony-counter

It helps to top up the wells with PBS or medium to reduce the liquid meniscus interfering with the scanning.
TITLE: Factors associated with dementia-related stigma and associated domains in adolescents: A Systematic Review Protocol
AUTHORS: Mrs   Esra Hassan, Prof Naji Tabet, Nicolas  Farina

[DESCRIPTION]
Background: To develop evidence-based anti-stigma programmes for adolescents, underlying  factors that drive dementia-related stigma in adolescents need to be captured. However, it is  unclear which factors play a role in dementia attitude formation in adolescents. This is a  protocol for a systematic review that will seek to identify factors associated with dementia  attitudes in adolescents (10-18 years old).  

Methods: A systematic search strategy will be used to identify observational data in which  factors are associated with dementia-related stigma and associated domains (e.g. attitudes,  beliefs, perceptions). Systematic searches for articles will be conducted in the following  databases: PubMed, PsycInfo, Web of Science, SCOPUS and Open Access Theses and  Dissertations. Titles and abstracts will be screened with the assistance of machine learning  software (ASReview), prior to reviewing full texts. Following data extraction, quality  assessment of study and risk of bias will be assessed using the mixed methods appraisal tool  (MMAT). The protocol is written in line with the Preferred Reporting Items for Systematic  Reviews and Meta-analyses Protocol (PRISMA-P) guidelines. 

Results: Descriptive data of the studies will be reported in tables and summarised narratively and split into thematic headings guided by the results of the systematic review. Identifying  which factors are implicated in attitudes towards people living with dementia will allow  researchers to be better informed in which factors may be a useful target for more tailored and  effective dementia related anti-stigma-initiatives.  

Discussion: The findings from this systematic review will help guide future research to identify  groups that may be at the greatest risk of developing stigmatising attitudes and potential targets  for future intervention. 

[STEPS]