eamag/protocols-with-steps huggingface dataset:
TITLE: Quantification of the molar concentration of the  NGS libraries by qPCR
AUTHORS: Yoko Kato-Unoki

[STEPS]
?. Using the NGS library concentration obtained from the Agilent BioAnalyzer, dilute the libraries to 100 pg/μl with 10 mM Tris-HCl, pH 8.0. Then prepare a 1:10000 dilution of each diluted library (final concentration, 0.01 pg/μl) by conducting 10-fold dilution series using 10 mM Tris-HCl, pH 8.0.*If the library concentration is unknown, conduct a 10-fold dilution series to achieve an appropriate final dilution (Note: for a 1:3 diluted library, we conducted a 1:500,000 dilution with 10 mM Tris-HCl, pH 8.0).
?. Check the product size and presence of adaptor dimers in a 1-μl aliquot of the PCR products by conducting electrophoresis through a 3% agarose–TBE gel. For example, in the Fig. 1, the lanes marked "×" show contamination with adaptor dimer.
?. Further clean-up the library with Agencourt AMpure XP beads (Beckman Coulter), if the library contains adaptor dimer. Refer to the Agencourt AMpure XP beads manual for reagent amounts and clean-up procedures. Elute to the same final volume as the original library and re-quantify and check again (i.e, repeat steps 1–3).
?. Calculate the molar concentration of each libraries from the standard curve.
?. Prepare a reaction mix and performs qPCR in accordance with the manufacturer's manual of KAPA Library Quantification kit (KAPA Biosystems).
TITLE: Resting-state Functional Magnetic Resonance Imaging Under Fast and Fed States in Healthy Human Subjects and Gastroparetic Patients
AUTHORS: Kun-Han  Lu, Kristine Mosier, John Wo, Terry Powley

[DESCRIPTION]
This protocol described the steps to acquire and analyze resting-state functional magnetic resonance imaging data under fast and fed states in healthy human subjects and patients with gastroparesis. 

[STEPS]
SECTION: Subjects
1. Twenty healthy subjects (14 females; 6 males) and 15 gastroparetic patients (11 females; 4 males) participated in this study under research protocols approved by the Institutional Review Board at Purdue University and Indiana University School of Medicine. All healthy subjects did not have a prior diagnosis of neurological, psychiatric, or gastrointestinal (GI) disorders. All gastroparetic patients had documented delayed gastric emptying by a standardized 4-h gastric emptying scintigraphy test (Tougas et al., 2000). Written informed consent was obtained from all participants. Standard MRI exclusion criteria were applied. 
SECTION: Experiment design
2. Subjects were studied using a 3T Siemens Prisma MRI scanner in the morning after a 12-h overnight fast. During the fast, subjects were asked to avoid any alcohol, caffeine, or medication that could affect GI function. Every subject underwent 3 fMRI sessions. Every subject first had a 5-min, eyes-closed, resting-state fMRI scan under a fast state. Then, the subject was instructed to consume a 350g meal consisting of blended natural ingredients (128g firm tofu, 95g pineapple chunks, 57g pineapple juice, 32g blueberry, and 38g banana). Immediately after the meal, every subject had another 5-min, eyes-closed, resting-state fMRI scan under a fed state. About 1-hour post-meal, every subject had another 5-min, eyes-closed, resting-state fMRI scan under a fed state.
SECTION: MRI acquisition
3. T1 and T2-weighted MRI and fMRI data were acquired in a 3T MRI system (Siemens, Magnetom Prisma, Germany) with a 64-channel receive-only phased-array head/neck coil. The fMRI data were acquired with 2 mm isotropic spatial resolution and 1s temporal resolution by using a gradient-recalled echo-planar imaging sequence (multiband = 8, 72 interleaved axial slices, TR = 1000 ms, TE = 31 ms, flip angle = 52°, field-of-view = 21 cm × 21 cm). MRI/fMRI images were preprocessed using a similar pipeline as in the Human Connectome Project (Glasser et al., 2013). Briefly, all fMRI images were corrected for motion and slice timing, aligned to structural images, and normalized to the Montreal Neurological Institute (MNI) space.
TITLE: RNA Direct Lysis Method
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">RNA Direct Lysis method</div><div class = "text-block">(from Masse and Gottesman. 2003 Genes and Development. 17:2374-2383) (notes and italics are from E. Fozo by Selene Hess)</div></div>

[STEPS]
?. [Steps]
Grow cells to desired OD.
?. [Steps]
At desired OD, remove 750 ml of cells to a tube at 65°C containing 500 ml acid:phenol-chloroform with 100 ml of direct lysis solution (SET-UP at least 25 minutes prior to harvesting to ensure the components are at 65°C). Vortex to mix, and place the tube back at 65°C for 10-15 minutes.
Can store at -80 indefinitely. Once move on, can’t stop till step 7, ethanol.
?. [Steps]
Spin 13000 r.p.m., room temperature for 5-10 minutes.
(15-20 minutes if from -80 storage)
?. [Steps]
Transfer supernatant to a new tube containing 500 ml of 65°C acid phenol: chloroform. Spin as in step 3 (RT).
?. [Steps]
Transfer supernatant and extract with 400 ml “regular” phenol: chloroform. Gentle vortex. Spin as in step 3 (RT).  May need to do additional extractions at this point.
?. [Steps]
Transfer supernatant to a new tube and extract with 400 ml chloroform. Spin as in step 3 (RT).
N.B. Liz and Aixia are the only ones who do this step, so it is clearly optional!  In interface is thick, white goo.
?. [Steps]
Transfer supernatant and add 700 ml 99% ethanol (95% is fine). Place in -80 C for 20 minutes (or indefinitely).
?. [Steps]
Spin 13000 r.p.m. 4°C, 30 minutes. Wash with 400uL 70% ethanol. Let pellet air dry.
?. [Steps]
Resuspend in RNase free water (20-25uL).
?. [Steps]
Let sit for 10 minutes at RT and finish resuspending by pipetting up and down/flicking tube.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The following procedure provides guidelines for isolating cells from whole blood using <a href="https://www.stemcell.com/products/brands/rosettesep.html?utm_source=protocolsio&amp;utm_medium=referral" target="_blank" rel="noopener noreferrer">RosetteSep™</a>, an immunodensity cell separation platform that isolates specific cell subsets during density gradient centrifugation. RosetteSep™ first crosslinks unwanted cells to red blood cells (RBCs) present in the sample to form immunorosettes. When the sample is centrifuged over a density gradient medium (e.g. Lymphoprep™), the immunorosettes pellet, leaving highly purified cells at the interface between the plasma and the density gradient medium.</p>

[BEFORE_START]
<p>This is a general procedure; specific conditions may vary according to the cell type being enriched. Find RosetteSep™ protocols for a specific cell type and more information by clicking the link below.</p>
<p> </p>
<p><a href="https://www.stemcell.com/products/brands/rosettesep.html?utm_source=protocolsio&amp;utm_medium=referral" target="_blank" rel="noopener noreferrer">https://www.stemcell.com/products/brands/rosettesep.html</a></p>
<p> </p>

[GUIDELINES]
<p> </p>
<h3><em>High-Throughput Protocol for NK Cell and Lymphocyte Isolation</em></h3>
<p>Traditional methods for isolating lymphocytes from blood are laborious and time-consuming, requiring precision and technical expertise. These methods typically involve isolating peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation before enriching specific cell subpopulations using immunomagnetic column-based systems. Using these conventional methods, isolating highly purified natural killer (NK) cells from a 450 mL unit of blood is a lengthy process (taking up to 4 hours), and it is difficult to process multiple samples quickly and efficiently.<sup>1</sup></p>
<p> </p>
<p>The new RosetteSep™ and SepMate™ system allow faster and more efficient blood processing without compromising cell function or performance in downstream assays. At the University of Maryland School of Medicine, Dr. Ajay Jain and colleagues routinely isolate NK cells from large numbers of human samples. In order to streamline their workflow and achieve higher-throughput sample processing, Jain’s lab has adopted the new RosetteSep™ and Sepmate™ cell isolation system in place of their previous, column-based method.</p>
<p> </p>
<p>See this Journal of Immunological Methods <a href="http://www.sciencedirect.com/science/article/pii/S0022175913001488" target="_blank" rel="noopener noreferrer">paper</a><sup>1</sup> for the detailed protocol.</p>
<p> </p>
<ul>
<li>1. So E.C. et al., J. Immunol. Methods. 2013. http://dx.doi.org/10.1016/j.jim.2013.05.001.</li>
</ul>
<p> </p>
<div>Have a question about this <a href="https://www.stemcell.com/isolating-specific-cell-subsets-directly-from-whole-blood-without-columns-or-magnets.html?utm_source=protocolsio&amp;utm_medium=referral" target="_blank" rel="noopener noreferrer">protocol</a>? Ask us by sending an email to techsupport@stemcell.com.</div>

[STEPS]
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?.
TITLE: Tris Buffered Saline (TBS)
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]
?. [Tris Buffered Saline (TBS)]
Prepare  by dissolving  and  in .
10 mM Tris150 mM NaCl
[of TBS]
[Tris Base]
[NaCl]
[of distilled water]
?. [Tris Buffered Saline (TBS)]
Adjust pH before use.
Tris has a pKa of 8.3. Hence, the buffering capacity at   is minimal compared to phosphate buffer (pKa = 7.21)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in "<a href="https://www.protocols.io/view/Obtaining-pure-cyanophage-stocks-liquid-assay-dqm5u5" target="_blank">Obtaining pure cyanophage stock: plaque purification</a>"

[GUIDELINES]
Use either the <a href="https://www.protocols.io/view/Liquid-Amplification-dq65zd" target="_blank">liquid amplification method (A)</a> or the plate amplification method (B) in the "<a href="https://www.protocols.io/view/Obtaining-pure-cyanophage-stocks-liquid-assay-dqm5u5" target="_blank">Obtaining pure cyanophage stock: plaque purification</a>" protocol.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Single action sequence reinforcement 
AUTHORS: Jonathan Tang

[DESCRIPTION]
Single action sequence reinforcement protocol for mouse studies from Tang et al 2023. 

[GUIDELINES]
Individual mice were subjected to a single session of protocol each day, with sessions following each other on consecutive days. 

[STEPS]
SECTION: Session 1 + Baseline
1. Mice were placed in a white open field box for closed loop reinforcement protocol. 
To acquire baseline behavior, individual mice were allowed to behave freely inside the box for 30 minutes on the first action A reinforcement session.
2. After initial behavior acquisition, closed loop reinforcement was initated by blue laser stimulation of VTA DA neurons were made available for 60 minutes.
SECTION: Sessions 2 &amp; 3
3. 90 minutes of closed loop reinforcement were made available for individual mice during sessions.
SECTION: Session 4
4. An extinction protocol was carried out comprising of 20-minute maintenance of reinforced behavior with laser availability, followed by 60 minutes of extinction of reinforced behavior without laser availability, followed by 20-minute re-acquisition of reinforced behavior with laser availability. 
SECTION: Contingency Degradation Protocol
5. Upon completion of the reinforcement and extinction protocols for action B, a contingency degradation protocol was performed comprising of 20-minute maintenance of action B with laser availability, followed by 60 minutes of contingency degradation of reinforced behavior by triggering laser randomly, followed by 40-minute re-acquisition of reinforced behavior with laser availability for action B performance.
TITLE:  Organoid Drug Treatment
AUTHORS: yzhu

[DESCRIPTION]
Protocol for drug experiments of organoid samples

[STEPS]
1. Trypsinize organoids and plate at 30,000 cells/well in 24-well plates
2. Incubate 5-7 days
3. Incubate organoids in a complete medium containing 500 nM JQ1 for 24 hours (JQ cells) or 0.1% v/v DMSO (control cells)
4. Harvest organoids in Cell Recovery Solution (Corning) on ice for 1 hour, wash with PBS, and centrifuge to retrieve cell pellets
5. Proceed to process cell pellets for RNA extraction
TITLE: Integrated Extraction for Cells
AUTHORS: Juan  Sanchez

[DESCRIPTION]
This liquid-liquid extraction for cells enables thesimultaneous generation of lipidomics and metabolomics samples for HILIC and C18 chromatography.

[GUIDELINES]
This extraction should be performed in a fume hood.

[STEPS]
1. Cells should arrive pelleted and frozen from collaborator 
2. Using wide-mouth pipette wash pellet with 500uL 150 mM ammonium acetate (25x aspirations)
3. Centrifuge @ 200 g for 5 min
4. Remove supernatant
5. Repeat steps 2-4 a second time
6. Add 4-6 non-sterile 2.3mm stainless steel beads to the tube.
7. Dispense 225 ul of ice-cold Methanol at -20 deg C containing 1.5% iSTD “SPLASH” mix into extraction tube 1.
8. Seal the sample tube and place it in the bead beater at an amplitude 20 Hz for 15 minutes to homogenize the sample & extract the metabolites.
9. Close the tube.
10. Vortex extraction tube 1 for 10 seconds.
11. Open extraction tube 1 and dispense 750ul of ice-cold MTBE.
12. Close extraction tube 1 and vortex for 10s.
13. Shake extraction tube 1 for 6 min at 4 deg C.
14. Open extraction tube 1 and dispense 188 ul of LC-MS grade water containing the 18 HILIC iSTDs at 1/3rd of their normal concentration as defined in the HILIC extraction SOP except CUDA.
15. Close extraction tube 1 and vortex it for 20 seconds
16. Centrifuge extraction tube 1 for 2 min at 14,000xg.
17. Open extraction tube 1 and transfer 700ul of the upper phase into a single 700ul tubetwo new separate tubes(350ul/ tube);  henceforth referred to as (Lipidomics tube 1) and (Lipidomics tube 2).
18. Transfer 250ul of the bottom phase from extraction tube 1 into a new single-tube hereafter referred to as (Hilic tube 1).
19. Centrivap Lipidomics tube 1, and Hilic tube 1 at room temperature until dry.
20. Reconstitute Lipidomics tube 1 in 120 ul of Methanol containing CUDA istd and close the tube.
21. Perform lipid cleanup on HILIC tube 1:  Add 500uL of 1:1 ACN:Water and vortex for 20s
22. Centrifuge at 14,000 g for 2 min.
23. Transfer supernatant to a new tube and dry down
24. Reconstitute HILIC tube 1 in 60ul of ACN:H2O (4:1 v/v) containing CUDA and close the tube.
25. Sonicate Lipidomics tube 1 and Hilic tube 1 at room temperature for 5 min.
26. Centrifuge Lipidomics tube 1 and Hilic tube 1 at 14,000xg for 2 min.
27. Open lipidomics tube 1, transfer equal fractions of the extract into two separate amber glass vials with micro-inserts, hereafter referred to as (lipidomics vial 1 pos) and (lipidomics vial 1 neg), and close the vials.a
28. Open Hilic tube 1 and transfer the extract into an amber glass vial with a micro-insert, hereafter referred to as (Hilic vial) and close the vial.
29. Analyze the samples via UHPLC–Orbitrap as follows.
TITLE: Design and preparation of synthetic reference peptides for APP/Aβ TOMAHAQ proteomics
AUTHORS: Hankum Park, Frances V Hundley, Harper JW

[DESCRIPTION]
TOMAHAQ-based targeted proteomics relies on heavy-labeled reference peptides for multi-plexed quantification of peptides of interest within a set of samples. The APP amyloid precursor protein is thought to be proteolytically processed within the endolysosomal system by β-secretase and ϒ-secretase to yield various forms of Aβ. To quantitatively track APP products, we describe a protocol for design and generation of synthetic reference peptides for TOMAHAQ analysis. We also include reference peptides for the extracellular and cytosolic domains.  

[STEPS]
1. Design APP peptides corresponding to extracellular and cytosolic regions based on data available in Peptide Atlas which are predicted to have favorable LC-MS properties. See attached Table.
2. Design half-tryptic peptides based on the cleavage sites for BACE1 and ϒ-secretase. See attached table.
3. Order commercial synthesis of peptides from Biomatik and Thermo Fischer Scientific, or similar. 
4. Reconstitute the peptides with 3% acetonitrile/0.1% formic acid, and quantify the concentration using Pierce Quantitative Fluorometric Peptide Assay.
5. Mix 10 μL of 200 μM peptide in 200 mM EPPS buffer (pH 8.5) with 1 μL of 5 μg/μL super-heavy TMT reagent (TMTsh). Incubate at 25 °C for 1 hr then check the labeling efficiency by LC-MS.
6. Add extra TMTsh to under-labeled peptides until > 95% labeling on both N-terminus and lysine residues is reached.
7. Desalt the reaction mixture using C18 StageTip.
8. Resuspend the peptides in 5% acetonitrile/8% formic acid, and pool the peptides.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
For use in <a href="https://www.protocols.io/view/Generating-viral-metagenomes-from-the-coral-holobi-ejgbcjw" target="_blank">Generating viral metagenomes from the coral holobiont</a>.

[STEPS]
?. 
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?. 
?. 
?. 
?. 
?. 
?. 
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?.
TITLE: Fresh 4% Paraformaldehyde in PBS
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Freshly prepared 4% Paraformaldehyde in PBS is used to fix adult and developing mouse brains during the transcardial perfusion process, as well as for immersion fixation.  This solution can be used for no more than one week post-preparation date.</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: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how to perform DNA FISH on dissected tissue of Planococcus citri. While the aim of this experiment was to distinguish localisation of the P. citri endosymbionts in ovarian and bacteriome tissue, the protocol can be easily adjusted for other tissues or the use of different probes.</p>
<p>The probes used here were designed according to the works of von Dohlen et al. (Nature, 2001) and Lopez-Madrigal et al. (PLOS ONE, 2013).</p>
<p>In brief, tissues were isolated in 1xPBS and transferred to various fixatives. After fixation samples were dehydrated and pre-incubated in hybridisation buffer before adding probes. After hybridisation, samples were washed and counter stained with Hoechst. FInally, stained tissues were transferred to microscopy slides, briefly air-dried and mounted in FLuorSave Reagent (Millipore).</p>

[STEPS]
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?.
TITLE: SOC Medium
AUTHORS: Alba Balletbó

[STEPS]
?. Add SOC medium components in a 1 L autoclavable. SOC medium components (1 L):  AB1Tryptone2% w/v2Yeast Extract0.5% w/v3NaCl10 mM4KCl2.5 mM5MgCl2 · 6H2O10 mM6MgSO4 · 6H2O10 mM7Glucose20 mM
AB1Tryptone2% w/v2Yeast Extract0.5% w/v3NaCl10 mM4KCl2.5 mM5MgCl2 · 6H2O10 mM6MgSO4 · 6H2O10 mM7Glucose20 mM
?. Add 1 L of distilled water into the bottle.
?. Vigorously shake the bottle.
?. Place the bottle into the autoclave and sterilize.
TITLE: Processing of leaf spectra
AUTHORS: Anna Schweiger, Etienne Laliberté

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Steps for processing leaf spectra measured with an integrating sphere</div></div>

[STEPS]
?. This step is taken care of by the CABO leaf spectra processing pipeline: Absolute reflectance and transmittance are calculated based on the most recent reference panel calibration. Depending on leaf size, calculations for absolute reflectance and transmittance are based on either 17 (Protocol “Measuring large leaves with an integrating sphere”, dx.doi.org/10.17504/protocols.io.q56dy9e) or 29 measurements (Protocol “Measuring small/narrow leaves with an integrating sphere”, dx.doi.org/10.17504/protocols.io.p8pdrvn) per leaf. Calculations for small leaves are based on Noda, H. M., T. Motohka, K. Murakami, H. Muraoka, and K. N. Nasahara. 2013. Accurate measurement of optical properties of narrow leaves and conifer needles with a typical integrating sphere and spectroradiometer. Plant, Cell & Environment 36:1903– 1909. https://doi.org/10.1111/pce.12100
?. Load output .csv files with absolute reflectance and transmittance from the processing pipeline
?. Match data (from processing pipeline) and metadata (from Fulcrum App)
?. Remove repeated wavelengths at the sensor overlap regions
?. Linear interpolation to 1 nm
?. Optional: Average spectra per individual plant for transmittance and reflectance
?. Optional: Apply Savitzky Golay filter to each spectral region (VIS, NIR, SWIR1, SWIR2)
?. Optional: Trim wavelengths to 400-2400 nm
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This mixture is used for the <a href="https://www.protocols.io/view/DNA-Extraction-Protocol-c32yqd" target="_blank">DNA Extraction Protocol</a>.

[GUIDELINES]
I used 400 µl CsCl purified lysate = add 1 µl of ProtK 20 mg/ml stock (20µg) and 20 µl 10% SDS stock (0.5% final concentration).

[STEPS]
?. 
?.
TITLE: Preparation of M9 Media
AUTHORS: NUS iGEM

[STEPS]
?. Prepare 5x M9 salt, 1M calcium chloride and magnesium sulfate solutions respectively using the following reagents.
?. Add2% casamino
200 ml
100 ml
100 µl
2 ml
?. Top up with sterile deionized water to make a litre of M9 media.
TITLE: Apple Keto Gummies Australia Reviews (Australia Consumer) Scam or Legit? Read Price 2022, Side Effects &amp; Ingredients
AUTHORS: health  

[DESCRIPTION]
Apple Keto Gummies Australia are just accessible on web-based stages. You should visit to true sites of a few makers and medical care brands. The trustworthy brands likewise offer weighty limits and arrangements on such items. Sign in to the confirmed site of makers and submit a request soon to get energizing arrangements up to a restricted time. Continuously purchase these sorts of enhancements with a specialist's remedy and guarantee the nature of the item prior to purchasing.

Official Website - https://rvmac.net/apple-keto-gummies-australia-reviews/

[STEPS]
TITLE: Cryopreservation of tissues for primary cell culture and single cell sequencing
AUTHORS: Harikrishna Nakshatri

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the cryopreservation of tissues used for primary cell cultures and single cell sequencing.</div></div>

[STEPS]
?. [Freezing Protocol]
Collect tissues in the Cryoprotective Freezing Medium with ROCK inhibitor.
?. [Freezing Protocol]
Resuspend in   and  + .
[primary cell medium]
[cryoprotective freezing medium]
[ROCK inhibitor]
?. [Freezing Protocol]
Aliquot into cryogenic storage vials.
?. [Freezing Protocol]
Cells should be frozen slowly at .
[per minute]
This can be achieved using the CoolCell containers at a   to   freezer overnight, then transferring to liquid nitrogen storage.
?. [Freezing Protocol]
Place vials in CoolCell containers.
?. [Freezing Protocol]
Mince tissues into small pieces.