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

[DESCRIPTION]
<p>This procedure is optimized for the isolation of 10<sup>7</sup> to 2 x 10<sup>8</sup> cells per tube. If working with fewer than 10<sup>7</sup> cells, keep volumes as indicated for 10<sup>7</sup> cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.</p>
<p> </p>
<p><strong>Product description and procedure summary: </strong></p>
<p>The kit is designed for the isolation of human memory CD4 T cells from PBMC. Target cells are depleted by incubating your sample with the biotin antibody cocktail followed by incubation with magnetic Streptavidin Nanobeads. The magnetically labeled fraction is retained by the use of a magnetic separator. The untouched cells are collected. These are the cells of interest; do not discard the liquid. Some of the downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>
<p> </p>

[GUIDELINES]
<p><strong>Reagents and instruments required: </strong></p>
<p> </p>
<p>MojoSort™ Buffer (5X) (Cat. No. 480017)</p>
<p>MojoSort™ Magnet (Cat. No. 480019) or compatible magnetic separation system</p>
<p>Adjustable pipettes</p>
<p>70 μm filters (one per sample)</p>
<p>5 mL (12 x 75 mm) polystyrene tubes</p>
<p>Reagents for sample preparation Reagents and instruments (Flow cytometer) to determine yield and purity</p>

[STEPS]
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TITLE: Eosin Y
AUTHORS: joshdm

[DESCRIPTION]
Preparation of stock and dilution of   


[STEPS]
SECTION: Prepare 1% Eosin Y stock
1. Weigh 2 g  of   and add it to a 1000mL flask

 

SECTION: Prepare 1% Eosin Y stock
2. Add 40 mL  of ddwater and swirl to mix

SECTION: Prepare 1% Eosin Y stock
3. add 160 mL of 95% ethanol and swirl to mix

SECTION: Prepare 1% Eosin Y stock
4. add 1 mL of Glacial Acetic acid 

SECTION: Preparing working dilution of Eosin Y
5. Use 80% ethanol to dilute stock to the desired concentration of eosin.
TITLE: Human Knee Tissue Procurement, Resection and Multimodal Analyses 
AUTHORS: Merissa Olmer, hswahn, Martin Lotz

[DESCRIPTION]
This protocol describes the procurement of entire human knee joints, resection of joint tissues and allocation for multimodal analyses. The protocol is focused on entire knees that are recovered by Organ Procurement Organizations and parts of the protocol are also applicable for human joint tissues that are removed during surgery on the knee or other joints. For entire knees, samples of all joint tissues are resected to allow analysis of ‘the knee as an organ’. 

[STEPS]
1. 
2.
TITLE: Protocol to UCSC Genome Browser & BLAST 
AUTHORS:  

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this lab, students will work through BLAST and the UCSC Genome browser to find and analyze information about their genes of interest. </div></div>

[STEPS]
?. [UCSC Genome Browser]
Genome BrowserYou will now have the opportunity to utilize these tools yourself to investigate a gene that you find interesting. Use these tools to answer the questions below and then relay these in the Lab Results of this Lab Notebook.Find a gene of interest and type it onto the genome browser. Use the human GRCh37/hg19 Assembly. In your own words, give a brief description of this gene’s function.
?. [UCSC Genome Browser]
Look through the tracks given on the displays and give the name of track which is used to give gene expression for different tissue types. What tissue do we observe the highest expression of RNA? (use track other than NCBI RefSeq)
?. [UCSC Genome Browser]
What orthologs does this gene have? What is the significance of having these as an ortholog?
?. [UCSC Genome Browser]
What Biochemical and Signaling Pathways is this gene involved in? Again, what is the signficance?
?. [UCSC Genome Browser]
What diseases are associated with this gene?
?. [BLAST]
BLASTDo a BLAST search of a gene of interest.
?. [BLAST]
Look at the BLAST results. Give the:Top 5 scoresTop 5 species this gene is found inLowest E-Value (if any)
TITLE: 10x  Protocols: Chromium Single Cell/Nuclei Gene Expression Flex Fixation for sc/sn -- University of Minnesota  TMCs (CG000478 Rev A)
AUTHORS: 10x Genomics

[DESCRIPTION]
DOIs for dissociation protocols and 10x Genomics fixation for Chromium Single Cell Expression flex protocols. 

Protocol ID# (CG) and Revision letter provided here: 
    10x CG000478, Revision A -- Protocol for fixation of single cells and nuclei prior to applying 10x Genomics Chromium flex protocols. 

Note: These protocols may not be the current version offered by the company but were used to produce the specific datasets connected to them. Please review the company support websites for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
1. https://www.10xgenomics.com/products/single-cell-gene-expression-flex
2. 10x Protocol CG000478, Rev A (Fixation):
TITLE: Protocols from Sales et al. (2020) Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays
AUTHORS: Cristina Rodrigues Gabriel Sales, Anabela  Silva, Elizabete Carmo-Silva

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This collection of protocols includes four assays for measuring Rubisco activity that are used and discussed in the manuscript "Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and </span><span style = "vertical-align:super;">14</span><span>C-based assays" (Sales et al. 2020).</span></div><div class = "text-block">Three of the protocols are NADH-linked microtiter plate-based assays for Rubisco activity that use alternative coupling enzymes:</div><div class = "text-block">1. GAPDH-GlyPDH, glyceraldehyde-3-phosphate-dehydrogenase and glycerolphosphate-dehydrogenase;</div><div class = "text-block">2. PEPC-MDH, phosphoenolpyruvate-carboxylase and malate-dehydrogenase;</div><div class = "text-block">3. PK-LDH, pyruvate-kinase and lactate-dehydrogenase.</div><div class = "text-block"><span>The radiometric assay for Rubisco activity measures the incorporation of </span><span style = "vertical-align:super;">14</span><span>CO</span><span style = "vertical-align:sub;">2</span><span> into 3-phosphoglycerate (3-PGA).</span></div><div class = "text-block">The fifth protocol describes the purification of 2,3-bisphosphate-dependent phosphoglycerate mutase (dPGM), which is required for the NADH-linked microtiter plate-based assays 2 (PEPC-MDH) and 3 (PK-LDH).</div></div>

[STEPS]
TITLE: Field Collecting Protocol for  Macrofungi
AUTHORS: Andrew Wilson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Sam Mitchel Herbarium of Fungi documents the diversity of “macrofungi” in the Southern Rocky Mountain region. For the use of this collecting protocol, we use the definition of Mueller et al. (2007, Biodiversity Conservation 16:37-48) to define macrofungi as, “...distinguished by having spore-bearing structures visible to the naked eye (mushrooms, brackets, puffballs, false-truffles, cup fungi, etc.)” Macrofungal surveys and collections represent baseline data critical to understanding species diversity and distributions. To make macrofungal collections valuable for current and future research, both the physical specimen and all associated data must be of the highest quality. If we use flowering plants as an analogy, the macrofungal spore bearing structure (AKA basidiocarp or “mushroom”) is analogous to the fruit of a plant. Consequently, we tend to refer to the mushroom as the “fruit” of the fungus. The ‘business end’ of the fungus (ie. the leaves, stems, and roots) are in the form of myceliumwhich form underground or within the substrate the fungus is deriving nutrition from. Please note that this protocol focuses on the field collection process. In Appendix see Resources 1-2 for information about processing museum-quality specimens for fungal herbaria.The directions outlined in this protocol are designed to research quality mushroom collections. </div></div>

[STEPS]
?. Planning and scope of the collecting trip.
?. Preparing for the field.
?. Describing the survey location so that its physical location and habitat characteristics can be well understood by others.
?. Collecting a macrofungal specimen with all the necessary physical structures, associated descriptors, and pictures/tissue collections.
?. Take notes in the data and features described in steps 3 and 4 above.
?. Drying to achieve a high quality and lasting specimen.
TITLE: Electrode Internal Solution with Biocytin II
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP provides instructions to prepare Internal Solution II used for simultaneous patch clamp electrophysiology experiments to probe circuit connectivity.</div><div class = "text-block"><span style = "font-weight:bold;">Note: </span><span>Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: LC-MS/MS Analysis
AUTHORS: Shiyi Wang

[DESCRIPTION]
LC-MS/MS Analysis

[STEPS]
1. **Sample Injection** - Inject 3 μL of each sample into the MClass UPLC system (Waters Corp).
2. **Sample Trapping** - Trap the sample on a Symmetry C18 20 mm × 180 μm trapping column at a flow rate of 5 μL/min with a solvent composition of 99.9/0.1 v/v water/acetonitrile.
3. **Analytical Separation** - Perform analytical separation using a 1.8 μm Acquity HSS T3 C18 75 μm × 250 mm column (Waters Corp.). - Use a 90-minute linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min). - Maintain the column temperature at 55°C.
4. **Mass Spectrometry Data Collection** - Perform data collection on the Thermo Orbitrap Fusion Lumos mass spectrometer equipped with a FAIMSPro device via a nanoelectrospray ionization source. - Use three different compensation voltages (-40V, -60V, -80V).
5. **Full MS Scan** - Use data-dependent acquisition (DDA) mode for each compensation voltage (CV). - Perform full MS scan from m/z 375 – 1500 with a resolution of 120,000 (@ m/z 200) and a target AGC value of 4e5 ions. - Set the max fill time to 35 ms.
6. **MS/MS Scan** - Acquire MS/MS scans with HCD settings of 30% in the linear ion trap in “rapid” mode. - Use a target AGC value of 1e4. - Set the max fill time to 35 ms. - Employ a 20-second dynamic exclusion to increase the depth of coverage.
7. **Cycle Time** - Set the total cycle time for each CV to 0.66 seconds. - Set total cycle times to 2 seconds between like full MS scans.
8. **Total Analysis Time** - The total analysis cycle time for each sample injection is approximately 2 hours.
TITLE: JAX SCBL workflow for scRNA-seq of human placenta
AUTHORS: Diane Luo, Jessica Grassmann, Paul Gabriel, William F Flynn, Elise Courtois, Paul Robson

[DESCRIPTION]
Described here is the workflow used by the JAX Single Cell Biology lab to generate single cell transcriptomic libraries from human placenta samples collected at the UCSD HuBMAP Female Reproductive Tissue Mapping Center.

[STEPS]
SECTION: Tissue collection
1. Tissue is collected and processed as soon as possible after Cesarean section or vaginal delivery according to the following protocol:

Human Placenta Tissue Collection and Preservation Methods - UCSD Female Reproductive TMC

Tissue is then stored in MACS storage buffer for overnight shipping.
SECTION: Sample preparation
2. Upon receiving the sample, single cells are isolated through a digestion with cold active protease as described here:

Human endometrium and endometriosis tissue dissociation for single-cell RNA sequencing
SECTION: Library Generation
3. The resulting loading, running, and library construction is performed according to the appropriate 10x Genomics protocol:

JAX - 10x Genomics - NEXTGEM v3.1 - 3' Gene Expression
SECTION: Sequencing
4. We have typically targeted 100,000 read pairs/cell for human placenta sample, resulting in target library sizes of roughly 600M-2,000M read pairs.  These are multiplexed on a NovaSeq 6000 S4 lane using v1.5 reagents and the reverse complement workflow in a 28/8/0/90 or 28/8/8/90 configuration.
SECTION: Sample preparation
2.1. The viability and cell concentration for each sample is assessed via a Countess FL II or LUNA FX7 automated cell counter.
SECTION: Sample preparation
2.2. The single cell suspension is then loaded onto one lane of the appropriate 10x Genomics gene expression chip as follows:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>ELISA (enzyme-linked immunosorbent assay) is a plate-based assay technique designed for detecting and quantifying peptides, proteins, antibodies, and hormones. In an ELISA, an antigen must be immobilized to a solid surface and then complexed with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product. The most crucial element of the detection strategy is a highly specific antibody-antigen interaction.</p>
<p> </p>
<p>This protocol describes the steps for performing sandwich ELISA.</p>

[STEPS]
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TITLE: Test-mate (Model 400) erythrocyte acetylcholinesterase (AChE) test
AUTHORS: H.K. Jeevan Dhanarisi, Indika B. Gawarammana, Fahim Mohamed, Michael Eddleston

[STEPS]
?. [Erythrocyte AChE activity analysis]
Remove Test-MateTM  components from the carrying case and lay them out, within easy reach, in front of you on a flat surface.
?. Press the “Power” key. The display screen indicates a 15 seconds warm up count down. Write up the temperature on your sheet.
?. Press the “Mode” key until “AChE mode” is displayed.
?. Remove the plastic covering of one of the reagent wells of the microplate with the biopsy punch. Add three drops of reagent to the well and dissolve the reagent by aspirating it using the transfer pipette. Fill the transfer pipette with the dissolved reagent.
?. Press the “Test” key. The display reads “Add blood” followed by “Insert tube” and “Press Test”. Add three drops of buffer to the tub e, mix with the stirring paddle.
?. Insert the tube into the colorimeter. Press the “Test” key. A 10 second “Blanking” is displayed ending in a “beep”. “Remove tube” followed by “Press Test” is displayed.
?. Press the “Test” key. The display reads “Add blood” followed by “Insert tube” and “Press Test”
?. Fill the capillary tube with blood (by sticking the end of the cleaned finger with the lancet) and dispense into the buffer filled tube after removing any excess blood from the outside of the capillary by rolling the end of the capillary over a filter paper. Shake the assay tube vigorously for 15 seconds and insert the tube into the sample analyzer.
?. Press the “Test” key. A 30 second “Reading” is displayed followed by a “beep”. During this time Hgb has been determined. The display reads “Remove tube” followed by “Press test”.
?. Press the “Test” key. The display reads “AT BEEP” followed by “Add reagent”. At the “beef” add the reagent in the transfer pipette to the diluted blood-filled tube. Insert tube followed by “Press test”. The display reads “Shake”. Shake the tubes by inverting five times.
?. Press the “Test” key. The display shows pre-incubation countdown followed by a 50 seconds reading phase ending in a “beep”. The analysis of erythrocyte AChE is concluded.The display reads “Remove tube” followed by “Press test”. Remove the tube and pour the solution into the waste bottle.
?. Press the “Test” key to initiate results recovery. After the first result is displayed, keep pressing the “Disp.” key to recover the rest of the data.
TITLE: Beetle rearing media
AUTHORS:   Tina, Yin-Tse    Huang

[DESCRIPTION]
ambrosia beetle rearing


[STEPS]
2. First, add dry ingredients
 

6. Put a double-layered cotton gauze on the mouth of the filled tubes. The cap is pre-drilled with holes that allow aeration and prevent contamination.
  

7. Autoclave the filled tube at 121 °C for 30 min.

8. Put autoclved tubes in an oven at 55°C for two days. (cotton gauze and cap remains on).
  

3. Second, add wet ingredients 
 

1. The recipe is for filling c.a. 18⁠—20 falcon tubes (⁠50ml).
5. To compress the media, use a clean 15ml falcon tube with a cap on, and press the media until it gets compressed.
4. Mix all the ingredients thoroughly and fill in 50ml falcon tubes. 
9. Happy beetle rearing!
TITLE: Single strain culture DNA extractions with polyzyme
AUTHORS: Jason Laurich, o.pogoutse, Megan Frederickson

[DESCRIPTION]
Protocol for DNA extractions from single strain bacterial cultures using the MetaPolyzyme Multilytic Enzyme mix (SIgma)

[STEPS]
SECTION: Bacterial culturing
1. - Place cells from agarose plates with single strain growing on it (preferably from single colony) into liquid culture. 

- Culture cells at appropriate temperature (27-30 C traditionally) for 1-4 days, as appropriate.
SECTION: Pelleting cells
2. - Centrifuge 1 mL of liquid bacterial culture for 2 minutes at 10,000 RPM in a 1.5+ mL Eppendorf tube. 

- If concerned about the amount of cells being too low, you can repeat this a second time by discarding supernatant, and then adding in 1mL of extra culture and repeating the process.

- This can be repeated again, though at a certain point, consider re-culturing strains to generate denser cultures.
SECTION: PBS wash
3. - Discard supernatant, and resuspend cells in 1 mL PBS and vortex. 

- Centrifuge and discard supernatant.

- Repeat above 2 steps.

- Repeat first 2 steps again (3 PBS washes total).

- Discard supernatant, and resuspend pellet in 150 µL PBS and vortex.
SECTION: Polyzyme digestion
4. - Add 2 µL of Polyzyme solution.

- Incubate at 35 C for 4-24 hours.
SECTION: Cell lysis
5. - Add 600 µL of lysis solution.

- Pipette up and down to suspend cells.

- Heat at (up to) 80 C for (up to) 10 minutes. Especially if samples are dense or cloudy. 
SECTION: RNAase treatment
6. - Add 1.2 µL of 10 mg/mL RNase A.

- Invert gently, ~ 2-5 times.

- Incubate at 37 C for 1 hour.
SECTION: Protein precipitation
7. - Cool to room temperature.

- Add 250 µL of protein precipitation solution.

- Invert gently, ~ 2-5 times to mix.

- Place on ice for 5 minutes.

- Centrifuge at 10,000 RPM for 10 minutes. 

- Transfer as much supernatant as possible (~900 µL works) to fresh sterile tubes using pipettes with tips removed.

- Repeat the protein precipitation steps (addition of solution through centrifugation) again.
SECTION: DNA precipitation
8. - Transfer 900 µL to 2 mL Eppendorf tubes with pre-aliquoted 600 µL 100% isopropanol. Use pipettes with tips removed.

- Invert gently 50 times!

- Pellet DNA at 13,000 RPM for 2 minutes.

- Discard isopropanol supernatant.

- Wash DNA with 1 mL 70% ethanol.

- Centrifuge at 13,000 RPM for 2 minutes.

- Pour off as much ethanol as possible. Be careful, the pellet may be loose!

- Air dry pellet for at least 15 minutes. This can be done in the safety cabinet, by placing the opening of the tubes over the air flow beneath the glass barrier. 

- Rehydrate DNA in ~25 µL (depending on volume of DNA, can be increased) Elution buffer (Qiagen) or TE buffer. Use pipettes with tips removed to suspend DNA by pipetting up and down.

SECTION: Measuring DNA
9. - Measure DNA concentration using Qubit or other instrument
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The protocol outlines how to aliquot PCR Master Mix for 5µL miniaturized amplification of 16S gene in 384-well format using the epMotion.

[BEFORE_START]
Please wear at least the minimum required personal protective equipment.
Ensure that all necessary kit components are available as well as user-supplied consumables.
Remove nuclease and nucleotide contamination from work surfaces and instruments prior to starting using an appropriate solution, such as RNase AWAY™ (Thermo Scientific™ catalogue: 700511), followed by wiping with 70% to 100% molecular biology grade ethanol to remove additional contaminants.

[STEPS]
SECTION: Mix reagents
?. 
SECTION: Setup epMotion automation platform
?. 
SECTION: Execute automated protocol
?.
TITLE: Collection of human vagal tissue samples for TEM imaging
AUTHORS: Terry  Powley, Deborah  Jaffey, Anita Gupta, Plamen Mihaylov, John Wo

[DESCRIPTION]
This protocol describes the methods used to generate samples of human vagus tissue suitable for TEM imaging.

[STEPS]
SECTION: Sampling
1. Vagus nerve samples were obtained intra-operatively from organ donors. Separate consents for organ donation and research were obtained. Informed consent for study participation was obtained from a parent and/or legal guardian. No organ donors were prisoners. A diagnosis of brain death or circulatory death as appropriate had been previously established by independent medical professionals according to local institutional regulations. All procedures related to tissue procurement and research use for tissues were in compliance with and approved by regulatory oversight review committees at University of Indiana School of Medicine, Indiana Donor Network or the local Organ Procurement Organization, and Purdue University. The vagus nerve biopsies were obtained from a continuous series of qualified organ donors meeting the diagnostic and medical criteria for organ donation and with consent for research participation in place. Donors from whom multivisceral transplants (lungs and stomach) were pursued were excluded from the research studies to enable removal of as complete sets of samples as possible. The research team was not involved with the identification and selection of organ donors.
SECTION: Sampling
3. After the completion of transplant organ harvesting, samples of the vagus nerve were collected. Approximately 10–20 mm long segments of the vagus nerve were removed from the following locations:
High thoracic trunk above pulmonary and cardiac branches – left / anterior
High thoracic trunk above pulmonary and cardiac branches – right / posterior
High abdominal trunk above hepatic and celiac branches – ventral / anterior
High abdominal trunk above celiac branches – dorsal / posterior
Ventral /anterior gastric branch at the level of the cardia
SECTION: Processing
4. All vagus nerve segments were immediately placed in a fixative solution containing 2% paraformaldehyde and 2.5% glutaraldehyde for 5 days at 4 °C.
  
  
The fixative solution was changed daily and the specimens were kept on an oscillating plate during the immersion fixation process (at 4 °C). The fix solution was always refrigerated and was always less than one week old. 
SECTION: Processing
5. After completion of the fixation process, the tissue specimens were rinsed (3 × 30 min at 4°C) in PBS, transferred to individual shipping vials under PBS, and shipped overnight on ice to the TEM laboratory for further processing and microscopy - see the protocol below:

 

SECTION: Sampling
2. Age, gender, BMI, race, cause of death, and delay time between death and sample recovery were recorded for all donors/samples. All data was deidentified.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Gen-IALFirst All-tissue DNA extraction kit -This protocol provides an efficient DNA extraction and purification of historic sample (tissue material)</p>

[BEFORE_START]
<p>Ancient DNA lab</p>

[STEPS]
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