eamag/protocols-with-steps huggingface dataset:
TITLE: Moore Swab Methods for Concentrating and Detecting Salmonella Typhi and Salmonella Paratyphi A in Environmental Wastewater Samples
AUTHORS: Pengbo   Liu;, Makoto   Ibaraki;, Christine Moe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This methods describes a tool to concentrate and testing Salmonella Typhi (ST) and Salmonella Paratyphi A (SPA) from environmental water samples (</span><span style = "font-style:italic;">e.g.</span><span>, wastewater, wastewater-impacted surface waters, etc.), using culture and/or molecular methods. This method should be applied in locations with adequate water flow so that a Moore swab can stay submerged across the sampling period. Results are reported as presence/absence.</span></div></div>

[STEPS]
?. [Summary of Moore Swab]
The Moore swab is placed into the source water for 24-72 hours. When water passes through the Moore swab, bacteria such as S. Typhi may be captured by size exclusion, on particulate matter, electrostatic interactions, or by other mechanisms. The sample is enriched in broth to recover the target microorganism, and the enrichment is extracted for molecular testing.
?. 
?. [Preparation]
2.1. UP broth2.2. Moore swab
?. Moore Swab Enrichment Procedures
?. [Preparation]
Procedures of Moore Swab Sample Collection
?. Real-Time PCR detection of Salmonella Typhi and Salmonella Paratyphi A
?. Membrane Filtration
?. DNA Extraction
TITLE: Making Freezing Solution
AUTHORS: Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The freezing solution can be made in advance and stored in sterile containers at room temperature till needed.</div></div>

[STEPS]
?. [Steps]
Book the autoclave beforehand
?. [Steps]
Make 0.05M K2HPO4 by dissolving 8.71g of K2HPO4 in 1L of DIW
?. [Steps]
Make 0.05M KH2PO4 by dissolving 6.80g of KH2PO4 in 1L of DIW
?. [Steps]
Make S Buffer solution by adding the following:129 ml 0.05 M K2HPO4871 ml 0.05 M KH2PO45.85 g NaCl
?. [Steps]
Make the freezing solution by adding the S buffer to 30% glycerol (v/v):Just add 700ml of S buffer to 300ml of glycerol to get the desired concentration
?. [Steps]
Autoclave the mixture. (Aliquoting first is not advised due to handling issues upon adding glycerol)
?. [Steps]
After autoclaving, aliquot into sterile 200ml bottles labelled with the name and date of the solution prepared.
?. [Notes]
Before using the freezing solution always check the bottle for any growth/contaminants
?. [Notes]
Discard the bottle if you see any contaminants and open a new one
?. [Notes]
Aliquot into falcon tubes from 200ml bottles to reduce contamination. Discard the tube after freezing do not put the contents back to the 200ml bottle!
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a target genomic site, but can also affect off-target sites. We develop a powerful assay for the unbiased identification of off-target sites that we term DISCOVER-seq. This approach takes advantage of the recruitment of endogenous DNA repair factors for genome-wide identification of Cas-induced double strand breaks. One such factor, MRE11, is recruited so precisely to a double stranded break that nuclease cut sites can be determined with single-base resolution. DISCOVER-seq is applicable to multiple types of Cas nucleases and provides an unprecedented molecular picture of events that precede repair of the affected sites. DISCOVER-seq furthermore detects off-targets in cellular models and tissues.

[STEPS]
SECTION: RNP editing of cells
?. 
SECTION: RNP editing of cells
?. 
SECTION: Crosslinking
?. 
SECTION: Crosslinking
?. 
SECTION: Crosslinking
?. 
SECTION: Crosslinking
?. 
SECTION: Crosslinking
?. 
SECTION: Cell lysis
?. 
SECTION: Cell lysis
?. 
SECTION: Cell lysis
?. 
SECTION: Cell lysis
?. 
SECTION: Cell lysis
?. 
SECTION: Cell lysis
?. 
SECTION: RNP editing of cells
?. 
SECTION: RNP editing of cells
?. 
SECTION: RNP editing of cells
?. 
SECTION: RNP editing of cells
?. 
SECTION: RNP editing of cells
?. 
SECTION: Cell lysis
?. 
SECTION: Cell lysis
?. 
SECTION: Prebinding of antibody to magnetic beads
?. 
SECTION: Prebinding of antibody to magnetic beads
?. 
SECTION: Prebinding of antibody to magnetic beads
?. 
SECTION: Prebinding of antibody to magnetic beads
?. 
SECTION: Prebinding of antibody to magnetic beads
?. 
SECTION: Chromatin IP
?. 
SECTION: Chromatin IP
?. 
SECTION: Wash beads and elution
?. 
SECTION: Wash beads and elution
?. 
SECTION: Wash beads and elution
?. 
SECTION: Wash beads and elution
?. 
SECTION: Wash beads and elution
?. 
SECTION: Wash beads and elution
?. 
SECTION: Wash beads and elution
?. 
SECTION: Wash beads and elution
?. 
SECTION: DNA purification
?. 
SECTION: DNA purification
?. 
SECTION: DNA purification
?. 
SECTION: DNA purification
?. 
SECTION: DNA purification
?. 
SECTION: DNA purification
?. 
SECTION: QC of pulldown
?. 
SECTION: Illumina Sequencing
?. 
SECTION: Amplicon-NGS for off-targets
?. 
SECTION: BLENDER bioinformatics pipeline
?.
TITLE: NLP screening (dot blot)
AUTHORS: Andreea S

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In order to determine if the His-tagged NLP is expressed and secreted by the bacteria, we plan to use the dot blot procedure with anti-His tag anti-bodies. Dot blot is an immunological technique used for detecting proteins directly from the culture supernatant (without gel separation). Thus, the samples are directly spotted on the membrane, making it a high throughput procedure ideal for testing different secretion signals. </div></div>

[STEPS]
?. On nitrocellulose membrane indicate the blotting region by drawing a grid by pencil.
?. Slowly applyof sample on the nitrocellulose membrane at the center of the grid.
2 µl
?. Use purified NLP-His and apply 5 points of increasing concentration of them on the nitrocellulose membrane in order to create a standard concentration curve.
?. Dry the membrane.
?. Block non-specific binding sites by washing the membrane in 5% BSA in TBS-T for 1h at room temperature.
?. Incubate with primary antibody diluted at the concentration recommended by the producer for 30 min. This step may require optimizing of the concentration.
?. Wash 3 times with TBS-T for
?. Incubate with the secondary antibody conjugated with HRP for (concentration recommended by the producer).
?. Wash 3 times with TBS-T (15 min, 5 min, 5 min)
?. Wash with TBS
?. Incubate with ECL reagent for 1 min. Develop the blot.
?. Compare the intensity of the sample with the standard curve in order to estimate the concentration.
TITLE: INDICAID Rapid Point-of-Care Test for SARS-CoV-2 Antigen
AUTHORS: Phase Diagnostics

[STEPS]
?. [Test Procedure]
The INDICAID Rapid Point-of-Care Test for SARS-CoV-2 Antigen includes swabs for nasal specimen collection.
?. [Test Procedure]
Remove the swab from the nostril and repeat on the other nostril using the same swab.
?. [Test Procedure]
The Collection Vial cap is composed of two parts. Remove the entire cap.
?. [Test Procedure]
Insert the swab so that it is completely submerged in the buffer.
?. [Test Procedure]
While submerged, twist the swab 20 times to release the specimen into the buffer.
?. [Test Procedure]
Discard the swab and close the cap on the Collection Vial tightly. Let it sit for   minute.
?. [Test Procedure]
Remove the small cap (top half only) from the Collection Vial to expose the dropper tip.
?. [Test Procedure]
Squeeze ALL contents of the Collection Vial into the Concentrating Vial.
?. [Test Procedure]
Close the cap of the Concentrating Vial and centrifuge the vial at
Centrifuge: 9000 33, 2 min
?. [Test Procedure]
After centrifuging, carefully open the Concentrating Vial and look for the clear bottom liquid layer. Fill the provided clean fixed volume pipette with    of the  clear bottom layer.
80 µl
?. [Test Procedure]
Apply all the contents of the pipette into the Test Cassette sample well.
?. [Test Procedure]
Read the result in the result window of the test device after   minutes.
?. [Test Procedure]
Insert the swab into one of the nostrils until a slight resistance is met (approx. 1 cm from edge of nostril). Roll the swab for  seconds inside the nostril to ensure that both cells and mucus are collected.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The consumption of antibiotics is a major driver for the development of antibiotic resistance in bacteria. We identified the trends and patterns of total antibiotic consumption in China’s tertiary hospitals from 2011 to 2015. We retrospectively analyzed aggregated monthly surveillance data on antibiotic sales made to 468 hospitals from 28 provinces. Antibiotic consumption was expressed in DDD per 1000 inhabitants per day (DID). We compared population weighted antibiotic consumption patterns in China with European countries using indicators from the European Surveillance of Antimicrobial Consumption (ESAC). Total antibiotic consumption in general, including all the specific antibiotic class except for aminoglycoside antibacterials, were significantly increased during the study period from an average of 7.97 DID in 2011 to 10.08 DID in 2015. In 2015, eastern regions consumed the most antibiotics if using population based calculation while west regions consumed the most using inpatient volume based calculation. Cephalosporins accounted for 28.6% of total DID, followed by beta-lactam-beta-lactamase inhibitor combinations with 20.0%, macrolides with 17.4%, and fluoroquinolones with 10.5%. Antibiotic in parenteral form were accounted for nearly half of all antibiotics. We used aggregated sales records data to monitor antibiotic consumption across China over a 6 year period. Although great efforts had been made through antibiotic stewardship in the past few years, total antibiotic consumption showed a significant upward trend during the study period. A consistent preferences for cephalosporins, macrolides, beta-lactam-beta-lactamase inhibitor combinations, as well as parenteral preparations was observed. More efforts are needed in the future to investigate the quality of antibiotic prescribing.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Automatic Deposition of CHCA Matrix  for MALDI Analysis of Lipids
AUTHORS: Elizabeth Neumann, Carrie  Romer, Jamie Allen, Jeff Spraggins

[DESCRIPTION]
Scope:
To describe the procedure for spraying tissue sections with CHCA for lipids.  CHCA is used as a safer matrix to ship coated slides to collaborators.

Expected Outcome:
Slides should be coated with CHCA.  Tissue sections should be imaged within 24 hours of matrix application.

[GUIDELINES]
Definitions:
 ACN is Acetonitrile
 MeOH is Methyl Alcohol/Methanol
 CHCA is α-cyano-4-hydroxycinnamic acid
 TFA is trifluoroacetic acid

[STEPS]
SECTION: Autofluorescence Scan
1. Remove slides from freezer and place in desiccator for 30 min .

SECTION: Autofluorescence Scan
2. Scan for autofluorescence on Zeiss Axio Scanner.
SECTION: TM Sprayer Setup
3. Change nitrogen to 6 psi and turn on sprayer.
SECTION: TM Sprayer Setup
4. Open software and set nozzle temperature to 85 °C 

SECTION: TM Sprayer Setup
5. Change LC solvent to 70% methanol at 0.1 mL/min.
SECTION: TM Sprayer Setup
6. “Load” sample loop with 5 mL  methanol.

SECTION: TM Sprayer Setup
7. Switch sprayer to “Inject” and spray for 2 minutes.
SECTION: TM Sprayer Setup
8.  “Load” 6 mL  of matrix solution into sprayer loop.

SECTION: TM Sprayer Setup
9. Switch sprayer to “Inject” and spray for ~1 minute.  Use slide to check that matrix is flowing.
SECTION: TM Sprayer Setup
10. Tape slide(s) to the top of the stage and adjust software scanning area.

SECTION: TM Sprayer Setup
11. Set method in software:
    700 mm/min nozzle velocity 
    2 mm track spacing
    0.1 mL/min flow rate
    CC Pattern
    2 L/min flow rate
    8 passes
    40mm nozzle height
    60 second drying time
SECTION: TM Sprayer Setup
12. Save method and make sure it is highlighted.
SECTION: TM Sprayer Setup
13. Under Cycle, click “Start,” then click “Continue.”
SECTION: TM Sprayer Setup
14. When finished, remove slide and place in slide box.
SECTION: Cleanup
15. Set TM sprayer temperature to 30 °C .

SECTION: Cleanup
16. Switch A1 solvent line to 100% ACN and set flow rate to 0.5 mL/min.
SECTION: Cleanup
17. “Load” loop with 20 mL  acetonitrile and “Inject” loop for 10 min .


SECTION: Cleanup
18. Spray tip of nozzle with MEOH.


SECTION: Cleanup
19. Log the pressure at your flow rate post cleaning. It must be within 3-4 psi of the starting pressure; if it is not, the cleaning procedure must be repeated.
SECTION: Cleanup
20. Change the HLPC flow rate to 0.05 mL/min.
SECTION: Cleanup
21. Spray nozzle with methanol.
SECTION: Cleanup
22. Remove the foil and wipe stage down with methanol.
SECTION: Cleanup
23. Replace wypall and bench diapers.
SECTION: Cleanup
24. Place all syringes in the biohazard container and empty the solvent waste.
SECTION: Cleanup
25. Shut down the software and turn off the TM Sprayer.
SECTION: Cleanup
26. Shut the nitrogen off.
TITLE: Semi-Structured Interview Protocol, Community Perceptions and Expectations of the Gump Station in Mo&#39;orea, French Polynesia
AUTHORS: Grace Sandel

[DESCRIPTION]
This project aims to understand community perceptions and expectations of the Gump Station in Mo’orea, French Polynesia and how the Gump Station can promote the co-creation of knowledge. The project will rely on qualitative research methods such as semi-structured interviews and workshops with community members across a variety of ages and backgrounds. This protocol is for conducting semi-structured interviews with participants. 


[STEPS]
SECTION: Interview questions
4. Where do you live? How long have you lived on Mo'orea for?
SECTION: Interview questions
6. What do you do for a living?
SECTION: Interview questions
8. Have you heard of the Gump Station?
SECTION: Interview questions
7. What comes to mind when you think about science and research on Mo'orea?
SECTION: Interview questions
9. Have you interacted with researchers from the Gump Station or other research stations like CRIOBE?
SECTION: Interview questions
9.1. [if participant answered yes] Can you describe one of these interactions?
SECTION: Interview questions
10. Have you heard of any current research projects on Mo'orea?
SECTION: Uploading audio file and notes
12. Save and name file "Interview Number, Date." Upload to shared Interview Recordings folder on Google Drive. 
SECTION: Beginning the interview
1. Ask for permission to record. If participant declines, explain that you will take notes with pen and paper. 
SECTION: Beginning the interview
2. If given permission to record, begin recording.

SECTION: Beginning the interview
3. Explain that the point of this interview is to get an understanding of how Mo'orea community members perceive research activities. Explain that the research project hopes to generate ideas about how the Gump Station can contribute to the community. 
SECTION: Interview questions
11. [Give brief descriptions of recent research projects at Gump Station, {projects to be selected}] Which of these projects would you like to hear the results from? Which of these do you think more people would be interested in learning more about or hearing the results from?
SECTION: Uploading audio file and notes
13. If notes were taken with pen and paper, transcribe them using a word processing system. Upload as a .docx file to the shared Interview Notes folder.
SECTION: Interview questions
5. What is your age?
SECTION: Interview questions
8.1. [if participant answered yes] What have you heard about the Gump Station?
SECTION: Interview questions
10.1. [if participant answered yes] What project(s) have you heard about? And do you think their research is useful or relevant?
TITLE: Immunofluorescence staining, vibratome sections
AUTHORS: eduard.bentea, María Sanchiz Calvo, Veerle Baekelandt

[DESCRIPTION]
Protocol for performing immunofluorescence staining on free-floating vibratome cut brain sections from rats or mice.

[STEPS]
SECTION: Day 1
1. Transfer sections to be stained to a new 24-well plate filled with 1X PBS. All incubations are with 500 µL solution per well unless stated otherwise.

SECTION: Day 1
3. Antigen retrieval step (using oven).
SECTION: Day 1
3.1. Incubate sections in antigen retrieval solution (10 mM citrate buffer pH 6.0 ; see recipe in Materials) at 80oC for 30 min.

SECTION: Day 1
2. 1X PBS rinse.
SECTION: Day 1
4. 1X PBS rinse.
SECTION: Day 1
3.2. Place the well plate in the same buffer on ice for 20 min.

SECTION: Day 1
5. Wash sections in 1X PBS for 5 min at room temperature on wobbler.

SECTION: Day 1
6. Wash sections in 1X PBS for 5 min at room temperature on wobbler.
SECTION: Day 1
7. Incubate sections with 250 µL blocking solution (PBS-T + 10% donkey serum) per well for 60 min at room temperature on wobbler. (PBS-T = PBS + 0.1% Tergitol)

SECTION: Day 1
8. Dilute primary antibodies at the required concentration in PBS-T + 10% donkey serum.
SECTION: Day 2
10. 1X PBS-T rinse.
SECTION: Day 1
9. Incubate sections with 250 µL primary antibodies overnight at room temperature.

SECTION: Day 2
11. Wash sections in 1X PBS-T for 5 min at room temperature on wobbler.
SECTION: Day 2
12. Wash sections in 1X PBS-T for 5 min at room temperature on wobbler.
SECTION: Day 2
13. Dilute secondary antibodies at the required concentration in PBS-T.
SECTION: Day 2
14. Incubate sections with 250 µL secondary antibodies for 120 min at room temperature (in the dark).

SECTION: Day 2
15. 1X PBS rinse.
SECTION: Day 2
16. Wash sections in 1X PBS for 5 min at room temperature on wobbler.
SECTION: Day 2
17. Wash sections in 1X PBS for 5 min at room temperature on wobbler.
SECTION: Day 2
18. Briefly rinse sections in 1/2 PBS + 1/2 AD and allow to dry.
SECTION: Day 2
19. Mount with Mowiol.
TITLE: AMOVA AND PHILOGENY IN HIV-1 PROTOCOLS
AUTHORS: Pierre Teodosio

[STEPS]
?. [METHODOLOGY]
?. [METHODOLOGY]
DATABANK: The 153 gene sequences of the integrase gene of human immunodeficiency virus 1 were collected from GENBANK (https://www.ncbi.nlm.nih.gov/popset/?term=MN888087.1 and participate in a PopSet dipped by Totmenin and collaborators on March 25, 2020 (Popset:1822236350).
?. [METHODOLOGY]
PHYLOGENETICS ANALYSES: For phylogenetic analyses, the previously described nucleotide sequences were used. The sequences were aligned using the MEGA X program (TAMURA et al., 2018) and gaps were extracted for the construction of phylogenetic trees.
?. [METHODOLOGY]
GENETIC STRUCTURING ANALYSES: Paired FST estimators were obtained with the software Arlequin v. 3.5 (EXCOFFIER et al., 2005) using 1000 random permutations. The FST matrix generated by the software was used in the construction of a dendrogram based on the UPGMA distance method with the MEGA X software (TAMURA et al., 2018) and the FST and geographic distance matrices were not compared.
TITLE: Preparing Agarose Gel
AUTHORS: alyssa.jeng

[DESCRIPTION]
N/A

[STEPS]
SECTION: Materials
1. 1X TAE Buffer
SYBRsafe
Agarose powder
Weigh boat
500mL Erlenmeyer flask
Gel Electrophoresis tray
Gel Electrophoresis comb
Tape
SECTION: Procedure
2. Tape up the gel electrophoresis tray and add well comb
SECTION: Procedure
3. Add 200mL of 1X TAE buffer to the Erlenmeyer flask
SECTION: Procedure
4. Weigh the right amount of agarose powder - depends on the expected PCR products: smaller than 500 bp: 2% agarose gel – 4 gr/200mL; bigger than 500 bp: 1% agarose gel – 2 gr/200mL).
SECTION: Procedure
5. Microwave for 2-3 minutes
SECTION: Procedure
6. Carefully remove flask from microwave and add 20uL of SYBRsafe to the gel solution. Swirl to mix.
SECTION: Procedure
7. Slowly pour the mixture in to the electrophoresis tray
SECTION: Procedure
8. Let cool for at least 30 minutes (no longer than 2 hours)
SECTION: Procedure
9. Clean the Erlenmeyer flask to remove excess agarose gel
TITLE: Ultrafiltration Methods for Concentrating and Detecting Salmonella Typhi and Salmonella Paratyphi A in Wastewater Samples 
AUTHORS: Pengbo  Liu, Makoto Ibaraki, Christine Moe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Tangential flow ultrafiltration is used to concentrate large volumes of environmental water samples (</span><span style = "font-style:italic;">e.g.</span><span>, wastewater, wastewater-impacted surface waters, etc.) for detection of </span><span style = "font-style:italic;">S. Typhi</span><span> and other microorganisms, using molecular methods. This method is applicable for sites with high flow rates, as a large volume is required. Results are quantifiable when enrichment is not used. </span></div><div class = "text-block"><span>This method was developed at Emory University, and it has been previously described in the following articles (Liu P et al, </span><span style = "font-style:italic;">J Microbiol Methods</span><span> 2012; Liu P et al, </span><span style = "font-style:italic;">J Appl Micobiology</span><span> 2013; Amin N et al</span><span style = "font-style:italic;">, Intern J Hygene Environ Health</span><span> 2020).</span></div><div class = "text-block"><span>A 40-L grab sample is collected from the source water using a bucket. The sample is brought to the lab and concentrated through a tangential flow ultrafilter. </span><span style = "font-style:italic;">S</span><span>. Typhi and S. Paratyphi A are captured on the filter by size exclusion, and eluted in ~500 ml volume of eluant buffer. The eluate is further concentrated by polyethylene (PEG) precipitation. The final pellet is extracted directly for molecular testing. </span></div></div>

[STEPS]
?. [Ultrafiltration Preparation]
?. [Ultrafiltration Procedures]
?. [Ultrafiltration Cleanup]
?. [PEG Precipitation]
?. [DNA Extraction]
?. [Real-time PCR Detection of Salmonella Typhi and Paratyphi]
TITLE: Thawing Cells
AUTHORS: salvatore  spanò

[DESCRIPTION]
Thawing cells, routine lab protocol

[STEPS]
SECTION: Thawing Cells
1. Take the cryovial from nitrogen, clean it with disinfectant, put it in the waterbath set to 37°C.
SECTION: Thawing Cells
2. In the meanwhile prepare a flask tagging it with cell line name, passage, date and operator. Fill it with medium 10ml.
If coating is required, check in advance which kind of is needed. Perform the coating accordingly to the related procedure and then perform step 2.
SECTION: Thawing Cells
3. Once vial is almost completely thawed, remove it from the waterbath. Prepare a falcon (cell line name, passage) with enough medium/Stop Solution  to wash the cells. 
Take the content of the cryovial and add it to the falcon. Wash the cryovial with 1 ml of medium/stopping solution.
Add it to the falcon.
SECTION: Thawing Cells
4. Centrifuge cells at 1200 RPM for 5 minutes. Remove pellet, resuspend cells accordingly to the number of flask to the seeded and cells number. thorougly pipette to prevent aggregation and seed cells in the flask.
SECTION: Thawing Cells
5.