eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for cleaning new and old teflon products.</p>

[GUIDELINES]
<p><span class="TextRun SCX158040763" lang="EN-US" style="font-size: 12pt;" xml:lang="EN-US"><span class="NormalTextRun SCX158040763" style="background-color: inherit;">All acid soaking is done HOT but not boiling on hotplate in clean lab (you may want to loosen nuts on jars to reduce pressure)</span></span></p>
<p> </p>
<p><strong><span class="TextRun SCX158040763" lang="EN-US" style="font-size: 12pt;" xml:lang="EN-US"><span class="NormalTextRun SCX158040763" style="background-color: inherit;"></span></span></strong></p>
<p>-50% acids should be recycled until you feel uncomfortable doing so, 1-3M can be (use your own intuition on how dirty it is)....weak acids are never recycled. </p>
<p> </p>
<p>-Teflon cleaning jars AND still acid bottles from beth have been handled with vinyl gloves, be careful not to handle them with clean PE gloves and then handle other things. </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>DNA is extracted from peripheral blood using QIAamp DNA mini kit (Qiagen) in accordance to the blood and body fluid protocol of the user manual supplied with the kit.</p>

[STEPS]
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TITLE: Overall protocol for top-down LC-MS/MS of human spleen tissue
AUTHORS: Jeannie  Camarillo, Bryon  Drown, Neil  Kelleher

[DESCRIPTION]
Overview description of the process for acquiring top-down LC-MS/MS data on human spleen tissue

[STEPS]
SECTION: Tissue Collection
1. Spleen tissue was provided by University of Florida TMC. Tissue was collected and prepared according to the following: 


 


SECTION: Sample Preparation
2. Perform LC based Proteomics on adjacent tissue sections:

 

SECTION: Data Acquisition
3. 


 

SECTION: Data Analysis
4.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a modification of the origianl Chelex 100 extraction described by Walsh, Metzger and Higuchi (1991 BioTechniques 10(4):506). Adding PVPP facilitates working with environmental samples as well as pure cultures hi in phenolics or other contaminants. </p>

[STEPS]
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TITLE: ChAT Immunofluorescent Staining
AUTHORS: Haley Geertsma

[DESCRIPTION]
This protocol is designed for 3-day choline acetyltransferase (ChAT) staining using the EMD Millipore AB144P (RRID:AB_2079751) antibody. Tissue stained with this protocol include 40μm free-floating mouse brain and spinal cord sections, and 20μm pre-mounted retina sections. All tissue was from mice perfused with 10% buffered formalin. 


[STEPS]
SECTION: ChAT Immunofluorescent Staining
1. Wash tissue slices in 1X phosphate buffered saline (PBS) for 5 minutes. Repeat x2.
SECTION: ChAT Immunofluorescent Staining
2. Incubate in blocking buffer (10% horse serum, 0.5% gelatin, 0.1% triton X-100 in 1X PBS) for 2 hours at room temperature.
SECTION: ChAT Immunofluorescent Staining
3. Incubate in primary antibody (goat anti-ChAT (RRID:AB_2079751) at 1:200 dilution in blocking buffer) at 4oC for 48 hours.
SECTION: ChAT Immunofluorescent Staining
4. Wash tissue with 1X PBS for 5 minutes. Repeat x4.
SECTION: ChAT Immunofluorescent Staining
5. Incubate in secondary antibody for 2 hours at room temperature.
SECTION: ChAT Immunofluorescent Staining
6. Wash tissue with 1X PBS for 5 minutes. Repeat x4.
SECTION: ChAT Immunofluorescent Staining
7. Mount free-floating sections on SuperFrost+ slides (if staining free-floating tissue) and let dry at room temperature for 15 minutes.
SECTION: ChAT Immunofluorescent Staining
8. Coverslip with fluorescent mounting medium and #1.5 coverslips. Outline coverslip with clear nail polish and store at 4oC or -20oC depending on length of storage.
TITLE: How the Metaverse will be used to preserve the culture of Tuvalu?
AUTHORS: Kalani Alcala

[DESCRIPTION]
To research the preservation of culture and sovereignty into a digital space I will be taking a social science approach. I plan on interviewing key actors, including but not limited to: government officials, technological leaders, cultural leaders, everyday residents. I also plan on sitting in on meetings to see how the culture is being digitized and uploaded to the metaverse. I will observe how the community is being engaged. I will collect data through recording meetings and interviews as well as careful note taking. Interviews will be somewhat standardized to gauge community understanding and feelings towards the process of digitization. In addition, I plan to count and categorize the different elements of culture that are being uploaded to the metaverse. This is a comprehensive research project which will cover many different aspects of the digital transformation of Tuvalu. 


[STEPS]
SECTION: Interview Protocol
1. Find people to interview. To do so, get in contact with community leaders and ask them to spread the word. 
- Make sure to offer compensation for people's time if possible. 
SECTION: Interview Protocol
2. Create a list of questions. 
 Tell a story about your favorite childhood memory in Tuvalu
Walk me through your experience with the Metaverse
How are you part of the metaverse creation?
What do you think about the metaverse and its potential to hold all Tuvaluan culture? 
What do you think is the most important aspect of Tuvalu? 
Etc
 
SECTION: Interview Protocol
3. Record Procedure with Audiotape.    
SECTION: Interview Protocol
4. Conduct interview with respect and good listening skills, prioritizing where the interviewee chooses to go and acknowledging them as the expert on the subject matter.  
SECTION: Meeting Observations
5. Attend local meetings that relate to the metaverse process of Tuvalu.
SECTION: Meeting Observations
6. Take notes and attempt to minimize presence. Remember you are acting as an observer, not a participant. 

SECTION: Meeting Observations
7. Analyze notes and see what you learned 
SECTION: Data Analysis of Tuvalu Uploads
8. When the Tuvalu metaverse has been created, explore the metaverse in a transect way. Write down in a spreadsheet the different forms of culture and brief description of how they have been digitally created.  Create spreadsheet.
TITLE: Adipose depot innervation: whole mount staining, imaging, quantification
AUTHORS: Jake Willows, Kristy Townsend, Magdalena Blaszkiewicz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Abstract</div><div class = "text-block">Little is known about the diversity and function of adipose tissue nerves due, in part, to the inability to effectively visualize the various nerve subtypes residing within these tissues. The tools currently available for researchers to image and quantify adipose tissue innervation are limited and dependent on optical clearing techniques and light sheet microscopy. Here we present a method of tissue processing that uses a method of mechanically compressing tissue to decrease tissue thickness in the z-axis by expanding it in the x and y-axes whilst leaving cells intact. This has been combined with autofluorescence quenching techniques to permit imaging of intact whole tissues on both widefield and confocal microscopes and a complementary means to perform whole tissue neurite density quantification. We have included examples of how this technique can be used to further our current knowledge of adipose-nerve communication by characterizing the nerves, nerve-subtypes, and neurovascular interactions within the inguinal subcutaneous white adipose tissue in mice.</div></div>

[STEPS]
?. See attachments under Abstract for PDF copies of protocols.
TITLE: True-Nuclear™ Transcription Factor Staining Protocol for 5 mL Tubes
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Perform cell surface staining as described in BioLegend's Cell Surface Immunofluorescence Staining Protocol.
?. Add 1mL of the True-Nuclear™ 1X Fix Concentrate to each tube, vortex and incubate at room temperature in the dark for 45-60 minutes. If necessary, the protocol can be suspended at this point. After discarding supernatant, re-suspend cells in CytoLast™ Buffer (Cat. No. 422501) or equivalent. Samples can be stored at 4°C for 12-18 hours, protected from light and plastic-wrapped to protect buffer evaporation.
?. Without washing, add 2mL of the True-Nuclear™ 1X Perm Bufferto each tube.
?. Centrifuge tubes at 300-400xg at room temperature for 5 minutes, and discard the supernatant.
?. Add 2mL of the True-Nuclear™ 1X Perm Buffer to each tube.
?. Centrifuge tubes at 300-400xg at room temperature for 5 minutes, and discard the supernatant.
?. Resuspend the cell pellet in 100µL of the True-Nuclear™ 1X Perm Buffer.
?. Add the appropriate amount of fluorochrome conjugated antibody diluted in True-Nuclear™ 1X Perm Buffer for detection of intracellular antigen(s) to each well and incubate in the dark at room temperature for at least 30 minutes.
?. Add 2mL of the True-Nuclear™ 1X Perm Buffer to each tube.
?. Centrifuge tubes at 300-400 x g at room temperature for 5 minutes, and discard the supernatant.
?. Add 2mL of cell staining buffer (Cat. No. 420201).
?. Centrifuge tubes at 300-400xg at room temperature for 5 minutes, and discard the supernatant.
?. Resuspend in 0.5mL cell staining buffer then acquire the tubes on a flow cytometer.
TITLE: Spatial Gene Expression for Formalin-Fixed Paraffin-Embedding (FFPE)-Lung Tissue using 10x Genomics Visium Spatial Transcriptomics
AUTHORS: Natalia-Del Pilar Vanegas, Lorena Rosas, Ana L. Mora, Mauricio Rojas

[DESCRIPTION]
We present a method to map gene expression in formalin-fixed paraffin-embedded (FFPE) lung tissue sections using the 10x Genomics Visium Spatial FFPE v1 protocol. This approach utilizes whole-transcriptome probes to achieve precise localization of mRNA within tissue structures. By preserving tissue morphology and ensuring reliable detection, this protocol enables comprehensive studies of lung biology and pathology. The spatial transcriptomics data obtained offer valuable insights into gene regulation and cellular diversity, enhancing our understanding of lung tissue organization and disease mechanisms.

[STEPS]
SECTION: Preservation of the samples:
1. Store formalin-fixed paraffin-embedded (FFPE) lung tissue blocks at 4 degrees Celsius to preserve RNA integrity.
SECTION: Tissue Quality Assessment:
2. RNA quality: High quality FFPE sections will have well-preserved RNA for accurate measurement. 
SECTION: Tissue Quality Assessment:
2.1. For Visium Spatial, the tissue preparation guide by 10x Genomics (CG000408, Rev D) recommends using the percentage of RNA fragments greater than 200 nucleotides (DV200) from extracted tissue sections for assessment.
SECTION: Tissue Quality Assessment:
2.2. Place three sections of 10μm thickness each in a 1.5ml microcentrifuge tube and proceed to RNA extraction using Qiagen RNeasy FFPE Kit (Cat no. 73504) and following the the steps outlined in the RNeasy Handbook (RNeasy Handbook).
SECTION: Tissue Quality Assessment:
2.3. Determine the DV200 value of your samples utilizing the Agilent 2100 bioanalyzer system.
SECTION: Tissue Quality Assessment:
3. Morphological integrity: Tissue sections should be free from excessive tearing, folding, or artifacts that could compromise spatial information.
SECTION: Tissue Quality Assessment:
4. Hematoxylin and eosin (H&E) staining: H&E staining allows visualization of cellular morphology and how it changes in response to disease, which can be used to interpret the spatial gene expression data.
SECTION: Sample preparation:
5. To trim the tissue section of ≤6.5 x 6.5 mm and a thickness 5 µm for sectioning onto the Visium Spatial Gene Expression Slide, follow the tissue preparation guide by 10x Genomics (CG000408, Rev D).
SECTION: Sample preparation:
6. Perform all deparaffinization, staining, imaging, and decrosslinking steps using the test slide according to the protocol described by the 10x Genomics (CG000409, Rev C).
SECTION: Visium spatial v1 FFPE library construction:
7. To perform probe hybridization, ligation, release and extension, and library construction, follow the user guide by 10x Genomics (CG000407, Rev D).
SECTION: Visium spatial v1 FFPE library construction:
8. After obtaining DNA libraries, follow the sequencing requirements of 10x Genomics (CG000407, Rev D).
TITLE: Vandy – Oral glucose tolerance test
AUTHORS: Louise Lantier

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">The glucose tolerance test measures the clearance of an oral glucose load from the body. It is used to detect disturbances in glucose metabolism that can be linked to diabetes or metabolic syndrome. Animals are fasted for 5 hours, fasted blood glucose levels are determined before a solution of glucose is administered by oral gavage. Subsequently, the blood glucose and plasma insulin levels are measured at different time points during the following 120 minutes. </div></div>

[STEPS]
?. Weigh the mice. For mice of differing fat mass, we recommend dosing the bolus of glucose on lean mass. This requires body composition.
?. Fast mice for 5 hours by transferring mice to individual cages or containers (7am to 12pm).
?. Prepare an experiment record sheet, sticks for glucose measurement and syringe for oral gavage of the Dextrose solution.
?. Calculate and record the volume of 20% Dextrose solution required for each individual mouse for gavage as follows: a. To inject 2g of dextrose/kg body mass, the volume of the glucose gavage is:                           20% Dextrose (μl) = 10 x body weight (g)
?. Cut the tip of the tail using clean surgical scissors. A small drop of blood (
?. Collect 40µL of whole blood in a tube for measurement of insulin. This is greatly facilitated by use of a collection tube such as Sarstedt’s Microvette® CB 300 (cat # 16.443.100 for Heparin or cat # 16.444.100 for EDTA). Spin the blood 1min at 13k rpm and collect plasma in a clean tube (20µL). Store plasma on ice.
?. Orally gavage the mouse with the appropriate volume of glucose solution, as previously determined (point 4) and start the timer.
?. The blood glucose levels are measured at 5, 10, 15, 20, 30, 45, 60, 90 and 120 minutes after glucose gavage. Samples for insulin determination (20µl plasma) are collected at 10, 30, 60 and 120min (Table 1). For each sample, start the bleeding again by removing the clot from the first incision, massage the tail if blood flow is inadequate. Place a small drop of blood on a new test strip. Results are recorded in the record sheet.
?. Ensure that further blood loss from the incision is minimal by briefly applying pressure to the incision after each measurement.
?. At the end of the experiment, mice are euthanized for tissue collection or placed in a clean cage with water and food available ad libitum for recovery. Monitor the mice carefully to observe any abnormal behavior. Plasma samples are stored in -20 or -80ºC freezer until analysis.
TITLE:  Long-range correlation  character Investigation of SARS-CoV-2 Genbanks
AUTHORS: Sid Ali Ouadfeul

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The main goal of this propotoc is to use the  1D Wavelet Transform Modulus Maxima lines (WTMM) method  to investigate the Long-Range Correlation (LRC) through the  estimation of the  the so-called Hurst exponent of isolate SARS-CoV-2 coronavirus RNA sequence, the Knucleotidic, Purine, Pyramidine, Ameno, Keto and GC DNA coding are used.</div></div>

[STEPS]
?. 1-Gathering SARS-Cov-2 Genbanks
?. DNA Coding using the Knucleotidic, Purine, Pyrimidine, Ameno, Keto and GC methods1) The Knucleotidic DNA coding: T=2, G=-2, A=1, C=-12) Purine coding A=G=1 , C=T=-1 3) Pyrimidine C=T=1, A=G=-1. 4) Ameno groupe: A=C=1, G=T=-1. 5) Keto coding G=T=1, A=C=-1. 6) GC coding G=C=1, A=T=-1.
?. Continuous Wavelet transform calculation using the complex Morelet Analyzing waveletS(t) is the Coded Genbank
?. Maxima of the Modulus of the Continous Wavelet Transform Calculation
?. Partition Function Calculation
?. Calculation of Spectra of exponents and Singularities Calculation
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?. Long-Range Correlation character Invertigation besed on the estmated Hurst Exponent
TITLE: Plant Sample Preparation for ICP-AES Analysis of Salt-Stressed Plants
AUTHORS: Maryam Rahmati Ishka

[DESCRIPTION]
This procedure outlines the preparation of plant samples exposed to salt stress. For plants grown under treatments other than salt, a special buffer is needed to remove surface chemicals during the washing step. This protocol is specifically for samples grown under salt stress, as salt can be easily removed with water.

[BEFORE_START]
n/a

[GUIDELINES]
n/a

[STEPS]
SECTION: Plant Sample Preparation for ICP-AES Analysis of Salt-Stressed Plants:
1. Record the fresh weight of roots and shoots separately for each seedling.
SECTION: Plant Sample Preparation for ICP-AES Analysis of Salt-Stressed Plants:
2. Quickly dip the roots and shoots in deionized sterile water for approximately 30 seconds.
SECTION: Plant Sample Preparation for ICP-AES Analysis of Salt-Stressed Plants:
3. Gently dry the samples with a paper towel.
SECTION: Plant Sample Preparation for ICP-AES Analysis of Salt-Stressed Plants:
4. Place the root and shoot samples separately in properly labeled envelopes.
SECTION: Plant Sample Preparation for ICP-AES Analysis of Salt-Stressed Plants:
5. Put all the envelopes in an oven at 80°C for two days.
SECTION: Plant Sample Preparation for ICP-AES Analysis of Salt-Stressed Plants:
6. Record the dry weight of the samples in ICP glass sample tubes.
TITLE: PEI Transfection 
AUTHORS: Nobody

[STEPS]
?. [Day 1]
Split cells into wells - see relevant protocol
?. [Day 2]
Make a solution of     - 500ng transfection DNA    - 2.5ul PET    - Serum Free Media (OptiMEM) up to 50ul
?. [Day 2]
Vortex solution twice for 5 secondsLeave solution to incubate in hood for 15 minutes Add 50ul of solution per well (500ul) Incubate for 24-48hrs
?. [Day 3 ]
View fluorescence under microscopeFix cells
TITLE: Total RNA Isolation from Bulk Tissue or Isolated Cells
AUTHORS: Brad Godfrey

[STEPS]
?. [Procedures]
Transfer biopsy in  cryotube (polypropylene) in  RNAlater
1.8 ml
1 ml
?. [Procedures]
Use color coding cap inserts to indicate biopsy origin
?. [Procedures]
Store at   until RNA extraction
-20 °C
?. [Procedures]
Perform microdissection of glomeruli / tubulointerstitium
?. [Procedures]
Store dissected tissue portion in less than  RNAlater solution and store at  until total RNA extraction
50 µl
-20 °C
?. [Procedures]
For total RNA/DNA extraction use “AllPrep DNA/RNA Micro Kit” (# 80284) from Qiagen following respective protocol. Use optional Beta-ME for glomerular compartment.Note: Elute RNA in H2O in nucleotide low-binding tubes (Eppendorf #022431021) and store at . Note: Keep first flow-through (.
16 µl
-80 °C
Quality and quantity were measured using Agilent Bioanalyzer 2100Elute RNA in 16uL
?. [cDNA Generation]
1.Store cDNA at
-20 °C
?. [cDNA Generation]
Use NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (rRNA depletion workflow)1.	Use NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (rRNA depletion workflow)
?. [NGS Library Preparation]
Use NovaSeq 6000 to sequence. (The University of Michigan Advanced Genomics Core)1.	Use NovaSeq 6000 to sequence. (The University of Michigan Advanced Genomics Core)
TITLE: Citrate Buffer
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.</div></div>

[STEPS]
?. [Citrate Buffer]
Mix citric acid and sodium citrate solutions in the proportions indicated and adjust the final volume to  with deionized water.  ABCDEFGHIJ1mL of Citric acid46.540.035.031.525.520.516.011.87.22mL of Sodium citrate3.51015.018.524.529.534.038.242.83pH3.03.43.84.24.65.05.45.86.2
pH range:   to  
(a) 0.1 M Citric acid: 19.21 g L-1 (M.W.: 192.1 g mol-1)(b) 0.1 M Sodium citrate dihydrate: 29.4 g/l (M.W.: 294.0 g mol-1)
100 ml
ABCDEFGHIJ1mL of Citric acid46.540.035.031.525.520.516.011.87.22mL of Sodium citrate3.51015.018.524.529.534.038.242.83pH3.03.43.84.24.65.05.45.86.2
?. [Citrate Buffer]
Adjust the final pH using a sensitive pH meter.
The use of pentahydrate salt of sodium citrate is not recommended.
TITLE: Lipoxygenase activity determination
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lipoxygenase activity on linoleic acid was determined according to the method described by Axelrod et al. (1981). This method determines the increase in absorbance at 234 nm, resulting from the formation of a conjugated double bond system in the formed hydroperoxide.</div></div>

[STEPS]
?. [Reagent Preparation]
10 mM sodium linoleate stock solution


In a 150 mL Erlenmeyer add:


10 mL distilled water (previously boiled)
78 μL linoleic acid
90 μL of tween 20

Keep the solution protected from light by wrapping the Erlenmeyer in aluminum foil.


Mix the solution with the aid of a pipette, taking care not to form bubbles.


Add 0.5 M NaOH until the solution is clarified (approximately 100 µL).


Transfer the solution to a 25 mL volumetric flask protected from light. Make up the volume to 25 mL.


Divide the stock solution of sodium linoleate into 1.5 mL amber microtubes and store at -20 ° C.
?. [Reagent Preparation]
50.0 mM phosphate buffer, pH 6.0Mix sodium phosphate monobasic and dibasic solutions in the proportions indicated below, and dilute to 200 mL with deionized water.6.15 mL of 0.2 M sodium phosphate, dibasic dihydrate (Na2HPO4•2H2O FW = 178.05)43.85 mL of 0.2M sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O FW = 138.01)Note: The dibasic stock sodium phosphate may be somewhat harder to dissolve; adding a little heat may help.Complete the final volume to 200 mL with deionized water.Adjust the final pH to 6.0.
?. [Procedure]
Pipette (in microliters) the following reagents into 1.5 mL microtubes  ABC1BlankTest2Phosphate Buffer1002 L1000 L3Sodium Linoleate Stock Solution10.0 L10.0 L4Enzymatic Extract (sample)-2.0 LMix by inversion
ABC1BlankTest2Phosphate Buffer1002 L1000 L3Sodium Linoleate Stock Solution10.0 L10.0 L4Enzymatic Extract (sample)-2.0 L
?. [Procedure]
Zero the spectrophotometer with Blank content at A234 nm.
?. [Procedure]
Immediately after enzyme addition (Test), mark the time and pour the contents into a suitable cuvette.


After 30 s of reaction onset, monitor readings at A234 nm for 120 s.
?. [Calculations]
Calculate velocity from absorbance values obtainedV0= ΔA234 nm (ε l Δt)-1V0: enzymatic activityΔA234 nm: absorbance variation at 234 nmε: molar extinction coefficient of linoleic acid hydroperoxides at 234 nml: optical pathΔt: time (120 s)
TITLE: Human induced pluripotent stem cell culture
AUTHORS: gurvir.virdi

[DESCRIPTION]
Cell culture for the maintenance of hiPSCs.

[STEPS]
1. Coat 6 well plates with Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific), 1:100 dilution for 1 hour before cell passaging. 
2. When hiPSCs are 80-90% confluent, they are passaged:
2.1. Old media is aspirated and replaced with 1ml of cell-culture PBS (Gibco, Thermo Fisher Scientific) 
2.2. PBS is aspirated and replaced with 0.5 mM EDTA (Thermo Fisher Scientific) made up in PBS. 
2.3. hiPSC are incubated in EDTA for 5 min at 37 °C .

2.4. After 5 minutes, the EDTA is aspirated and cells are dissociated with media (E8 or mTesR 1). 
2.5. hiPSCs are then placed in new 6 well plates at the desired dilution.
3. All hiPSCs are fed daily with fresh media. All cells were maintained at 37°C and 5% carbon dioxide.