eamag/protocols-with-steps huggingface dataset:
TITLE: Magnetic resonance imaging (MRI)
AUTHORS: Núria Peñuelas

[DESCRIPTION]
Magnetic resonance imaging (MRI) of mouse brain

[STEPS]
1. Prepare the equipment: 7T Bruker BioSpec 70/30USR scanner (Bruker BioSpin GmbH, Ettlingen, Germany) equipped with a mini-imaging gradient set (400mT/m) and using a 72-mm inner diameter linear volume coil as a transmitter and a dedicated mouse brain surface coil as a receiver. 
2. Prepare the software: Acquire and process all MRI data on a Linux computer using Paravision 5.1 software (Bruker BioSpin GmbH, Karlsruhe,Germany)(RRID:SCR_001964, http://www.bruker.com/service/support-upgrades/software-downloads/mri.html).
3. Anesthetize the animals (1.5-2% isoflurane in 1 L/min oxygen for maintenance) and place them into an animal bed with bite-bar and ear-bars for optimal head immobilization.
4. Control the respiration rate (50-100 bpm) and core body temperature (37 ± 1°C) with an animal monitoring and control system (SA Instruments, Stony Brook, NT).
5. Acquire high resolution T1-weighted spin-echo images in the axial plane with and without fat saturation pulses.

6. Acquire two slice packages of 7 contiguous slices each containing the SNpc and LC regions and using the following parameter: TE=7 ms; TR= 500 ms; NA=56; FOV=1.92×1.92 cm2; MTX=128×128; ST= 0.25 mm; thus, resulting in a spatial resolution of 150×150×250 µm3; Acqt= 1h per image.
TITLE: DQ-BSA and BMV109 microscopy
AUTHORS: Rebecca Wallings

[DESCRIPTION]
DQ-BSA and BMV109 microscopy for peritoneal macrophages

[STEPS]
1. BMV109 Pan Cathepsin probe (Vergent Bioscience) and DQ red BSA (Invitrogen) were added to each well at a final concentration of 1µM and 10µG/mL, respectively, and cells were incubated at
37°C for 1-hour. 

Cells were washed 3 x DPBS +/+ and fixed for 10 minutes at room temperature in Invitrogen‱ eBioscience‱ Intracellular Fixation buffer.

Cells were washed 3 x DPBS+/+ and incubated in 1 μg/ml DAPI (Life Technologies) for 10 minutes at room temperature in DPBS+/+.

 Cells were imaged using an EVOS‱ M7000 (Invitrogen) at 20 x magnification. Image analysis was performed using Cellprofiler 4.2.5.
TITLE: Adherent Cell Culture (generic)
AUTHORS: Gwen Phung, Philippa R Kennedy

[DESCRIPTION]
Standard procedure for culturing adherent cell lines. Follow these instructions and cell type-specific instructions on the Cell line information protocol

[BEFORE_START]
Warm complete media in 37oC water bath for at least 20 minutes to avoid shocking the cells. 
Sterilize the hood, ensure proper airflow. 
Wear a labcoat and gloves.

[GUIDELINES]
Maintain sterile culture technique throughout. 

Avoid reaching pipettes inside stock bottles or flasks - use a pipette gun and stripette to minimize the risk of contamination.
Avoid touching the neck of the flask with your pipette - this is a primary source of contamination. 
Avoid liquid touching the filter inside the flask and pipette gun. If they are touched, replace the pipette gun filter and flask lid.

When new to this technique, check your proficiency by growing cells without antibiotic. Infections will rapidly arise if there is a problem. Deep clean incubators, biological safety cabinets and pipette guns (swapping out their filters) if an infection arises to avoid it spreading.

Maintain a cell culture log with calculated doubling times to identify if there are changes to the growth pattern of your cells. Changes to growth patterns can indicate infections or inappropriate split ratios (cells reaching confluence too quickly or being seeded too sparsely).

[STEPS]
1. Remove spent media from flask. 
2. Add 5-10 mL of PBS. Gently swirl flask to rinse cells then remove PBS.
    - this removes serum proteins that might interfere with trypsin
3. Add trypsin (0.05%, Gibco Cat. No. 25300054)
1 mL for T25
2.5 mL for T75
5 mL for T150
4. Incubate flask at 37oC for 5-10 min. 
- check the flask at 5 min by holding it up to the light, if cells fall off by themselves, culture is ready, if some remain stuck, continue incubating up to 10 min
5. Add 4x volume of complete media into flask. Pipette gently up and down to collect dislodged cells. Transfer cells to a 15 mL conical tube. 
4 mL for T25
10 mL for T75
20 mL for T150
6. Centrifuge cells for 4 minutes at 300g.
7. Add appropriate volumes of complete media into a new flask. Label date, passage number and initials on flask. 
7 mL for T25
13 mL for T75
30 mL for T150
8. After centrifuging, remove supernatant. Break up cell pellet by tapping the tube. Resuspend cells with 1-2 mL of complete media. Transfer appropriate fraction of cell suspension into flask for the appropriate split ratio as indicated by 'Cell line information' protocol (dx.doi.org/10.17504/protocols.io.rm7vz82o8vx1/v2)
TITLE: Potassium phosphate buffer (0.9 M, pH 7.7)
AUTHORS: Andreas Sagen

[DESCRIPTION]
Potassium phosphate buffers are buffers with a buffering capacity between 5.8 and 8.0, where it is possible to adjust the pH and buffer strength with different ratios of Sodium phosphate monobasic and Sodium phosphate dibasic.

[STEPS]
SECTION: 500 mL Phosphate buffer (0.9 M, pH 7.7)
1. Fill a sterile 500 mL bottle with 400 mL distilled water
SECTION: 500 mL Phosphate buffer (0.9 M, pH 7.7)
2. Measure 62.705 g Potassium phosphate dibasic and 11.568 g Potassium phosphate monobasic

Materials:
 
 
SECTION: 500 mL Phosphate buffer (0.9 M, pH 7.7)
3. Add dry ingredients and mix for 5 min
SECTION: 500 mL Phosphate buffer (0.9 M, pH 7.7)
4. Fill to 500 mL with distilled water
SECTION: 500 mL Phosphate buffer (0.9 M, pH 7.7)
5. Filter sterilize through a 0.2 µm filter and store refrigerated (4 °C)
TITLE: 10x Protocols: Tissue Fixation &amp; Dissociation for Chromium Fixed RNA Profiling -- University of Minnesota TMCs (CG000553 Rev A)
AUTHORS: 10x Genomics

[DESCRIPTION]
10x Genomics protocol where you fix the whole tissue and then dissociate it in preparation for Chromium Single Cell Expression flex 

Protocol ID# (CG) and Revision letter provided here: 
    10x protocol CG000553, Revision A-- Fixation and dissociation of whole tissue; preparation for 10x Chromium Flex scRNAseq

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.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>Modified from Rioux, C., Jordan, D., &amp; Rattray, J. B. (1983). Colorimetric determination of catechol siderophores in microbial cultures. <em>Analytical Biochemistry,</em><em>133</em>(1), 163-169. doi:10.1016/0003-2697(83)90238-5</p>

[STEPS]
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TITLE: Inserting RFID into rats
AUTHORS: a.beeson  , Leah  Solsberg-Woods, Gabriel J  Barrero, Oksana  Polesskaya, Abraham  Palmer

[DESCRIPTION]
Insert RFID chips into rats

[GUIDELINES]
Save all old cage cards for verification of DOB. Weaned Pups will be housed in Coleman B113 for at least 2 days before transported to TSRB 155.

[STEPS]
2. Carefully install one RFID into the clean needle
3. scruff rat skin on the back of neck with fingers
4. Inset the sharp tips of the needle under the skin, target the back of the neck, close to the head.
5. Release the RFID by pushing the plunger of the syringe
6. Scan and make sure the RFID is under the skin

7. upload the scanned RFID into a spreadsheet
1. Disinfect needle with 70% EtOH and air dry
TITLE: Bacterial Isolation of Mycobacterium avium
AUTHORS: Tetsuya Komatsu, Kenji Ohya, Justice  Opare Odoi, Shota  Suganuma, Kotaro Sawai

[DESCRIPTION]
Mycobacterium avium subsp. hominissuis (MAH) is one of the most important agents causing non-tuberculosis mycobacterial infection in human and pigs. Genome analysis on MAH of human isolates has been proceeding, however, those of pigs are limited despite its potential source of infection to human. Here, we isolated  MAH from pig lymph nodes or livers and obtained their genomes.

[STEPS]
1. Cut out 20 mg of the caseous necrotic area and its surroundings from lymph nodes or organs and put them in a screw tube.
2. Put some 5 mm stainless steel spheres and 400 µl of 2% NaOH in the screw tube. 
3. Set the tube in a tabletop crusher and process it at 3,200 rpm for 30 seconds.
4. Add 400 µl of 0.5% N-Acetyl-L cysteine (NALC)-2% NaOH and allow to stand for 14 hours.
5. Inoculate 100 µl of the suspension into the 2% Ogawa PS medium and incubate at 37°C for 3 - 4 weeks.
TITLE: Histology, Immunohistochemistry and Imaging 
AUTHORS: Jonathan Tang

[DESCRIPTION]
Sample preparation, histology, immunohistochemistry (IHC) and imaging of brain sections in Tang et al 2023. 

[BEFORE_START]
Process begins after behavioral sessions were completed. 

[STEPS]
SECTION: Sample Preparation
1. Deeply anesthetized mouse with isoflurane. 
SECTION: Sample Preparation
2. Perfuse transcardially in PBS and then 4% PFA/PBS.
SECTION: Sample Preparation
3. Dissect brains with skulls attached and submerged in 4% PFA in PBS at 4 degrees Celsius overnight
SECTION: Sample Preparation
4. Next day-brains were rinsed 3 times in PBS. 
SECTION: Sample Preparation
5. Brain regions including VTA and implants were sectioned by vibratome into 50 or 100 μm slices (depending on desired output).
SECTION: Histology/Immunohistochemistry
6. Slices were stained with the following reagents by the Champalimaud Histopatholgy Platform (core facility) using their standard protocol. 
SECTION: Histology/Immunohistochemistry
6.1. -Rabbit anti-GFP 488 conjugate (1:1000;Molecular Probes A21311, RRID:AB_221477)
-Mouse Anti-TH (1:5000;Immunostar Th 22941, RRID:AB_572268) 
-Goat Anti-Mouse - IgG (H+L) Highly cross-adsorbed secondary antibody - Alexa Fluor647(1:1000; ThermoFisher, A-21236, RRID:AB_2535805)
-DAPI (1:1000 of 20 mg/mL stock; Sigma, D9542).
SECTION: Imaging
7. Using a Zeiss Axio Imager M2 microscope-10x tiled images were taken through the relevant fluorescent channels.
SECTION: Imaging
7.1. The M2 is equipped with a fast Colibri.7 LED illumination for excitation of fluorophores. Images are captured with a high-sensitivity monochromatic sCMOS camera(Hamamatsu Orca Flash 4.0 v2). The objective used for the images is a ZEISS Plan-ApoChromat 10x/0.45, which allowsto resolve up to 577 nm when using a wavelength of observation of 520nm and it is fully corrected for chromatic and spherical aberrations. Implant locations were determined using Paxinos and Franklin's the Mouse Brain in Stereotaxic Coordinates.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a Single-cell RNA-Seq expression analysis <em>Support Protocol </em>for <a href="https://www.protocols.io/view/preparation-of-single-cell-rna-seq-libraries-for-n-n6gdhbw?step=27" target="_blank" rel="noopener noreferrer">Preparation of Single-Cell RNA-Seq Libraries for Next Generation Sequencing</a>. </p>
<p> </p>
<p> </p>

[GUIDELINES]
<p>Following sequencing of the cDNA libraries on an Illumina sequencer, data is generated as a series of files in the FASTQ format. For each unique sample specified in the sequencing sample sheet, four files are generated: one containing the “left-hand” read data (one end of the paired-end reads), one containing the “right-hand” read data (the other end of the pair), one containing the “left-hand” Nextera indexing read data, and one containing the “right-hand” Nextera indexing read data. RNA-Seq analysis uses computational tools to match each read pair, align the read pair to the genome sequence, and quantify the number of reads that align within each annotated gene.</p>
<p> </p>
<p>The <a href="http://www.genomespace.org/" target="_blank" rel="noopener noreferrer">GenomeSpace web portal</a> was developed to assist researchers with minimal computational analysis experience. Using its drag-and-drop interface, data sets and modules of pre-built analytic tools can be organized into customizable pipelines for numerous applications. Despite its ease of use, GenomeSpace uses cloud storage and computing power, making it less efficient for a large number of sequencing analyses or if a researcher has access to higher computing power at their own institution; as an alternative strategy for higher throughput, we also provide a Unix-based workflow.</p>

[STEPS]
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TITLE: ABT263 (Navitoclax) Treatment in Mice
AUTHORS: Andrew Davis, Peter Adams

[DESCRIPTION]
This protocol is for treating mice with the senolytic, Navitoclax, by oral gavage.

[BEFORE_START]
ABT263 must first be dissolved in the vehicle by heating to 60°C.  Sonication can be used to dissolve faster. See the materials section for specifics.

[STEPS]
1. Give mice Navitoclax or Vehicle by oral gavage at 50mg/kg/day.  Mice receive vehicle or drug for a total of 14 days, spread across 6 weeks, as outlined in the sub-steps below.
1.1. Mice receive seven days of once-daily oral gavage of drug or vehicle
1.2. Then fourteen days with no treatment
1.3. Then seven more days of once-daily oral gavage of drug or vehicle
1.4. Then fourteen days with no treatment
1.5. Harvest tissues after the last day of no treatment
TITLE: Chemically Competent Cells
AUTHORS: Alba Balletbó

[STEPS]
?. Grow to OD600 0.6 in 0.1/10 mL LB.
?. Centrifuge at 8000rpm for 5 minutes.
?. Resuspend in 5mL ice-cold CaCl2.
?. Distribute 1.5 mL over 3 Eppendorfs and spin for 30 seconds, 8000rpm.
?. Resuspend pellet in 0.5 mL ice-cold CaCl2, flick the tube gently.
?. Aliquot into 50µL Cryo-tubes and store at -80ºC until transformation.
?. Culture desired strain O/N 0.5 mL/50 mL LB in a Greiner tube.
TITLE: Visualisation and quantification of dendritic spines in cultured human Medium Spiny Neurons (MSNs)
AUTHORS: Quyen Do

[DESCRIPTION]
This protocol describes the visualisation of dendritic spines of human neurons cultured on coverslips in vitro and subsequent quantification using the software Imaris.


[STEPS]
SECTION: Visualisation of fluorescent dendritic spines in MSNs
1. Mount a coverslip of Medium Spiny Neurons (MSNs) immunolabelled for DARP32 and neurobiotin onto SlowFade™ Diamond Antifade mountant (follow Protocol: Immunocytochemistry of cultured human Medium Spiny Neurons (MSNs)).

SECTION: Visualisation of fluorescent dendritic spines in MSNs
2. Place slide under under Olympus FluoView FV1000 confocal microscope with argon and solid-state laser with 488 nm and 559 nm excitation, respectively. 
SECTION: Visualisation of fluorescent dendritic spines in MSNs
3. Use the 60x oil-immersion objective (NA = 1.40) to image and capture MSNs coexpressing Darpp32 and Neurobiotin as Z-stacks, sampling sequentially at resolution of 1024 * 1024 pixels and at 1.05 µm steps, as optimised by Nyquist sampling theorem.
SECTION: Visualisation of fluorescent dendritic spines in MSNs
4. Use the zoom function (3x) in the Olympus Dendritic FluoView FV1000 software to capture dendritic branches of biotinylated neurons.


 


SECTION: Quantification of dendritic spines
5. Use the 'Surface' module to threshold and segment the confocal images.
SECTION: Quantification of dendritic spines
6. Use the 'Filament' module to automatically render the dendrites from images obtained after step 5.
SECTION: Quantification of dendritic spines
7. Choose the spine detection function in the 'Filament' module to detect spines as protrusions from the previously-identified dendritic filament. 
SECTION: Quantification of dendritic spines
8. Manually exclude putative spines at branch points or disconnected dots.
TITLE: Monoamine oxidase activity in fish brain tissue 
AUTHORS: Caio Maximino, Denis Broock Rosembeg

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol describes a spectrofluorophotometric method for rapid determination of monoamine oxidase (MAO) activity in zebrafish brains. The protocol is based on the transformation of kynuramine hydrobromide into 4-hydroxyquinoline. Since zebrafish possess only one MAO isoform, inhibitors of other isoforms are not necessary.</div></div>

[STEPS]
?. [Reagent setup]
Prepare tissue lysis buffer:16.8 mM Na2HPO4 and 10.6 mM KH2PO4,  , isotonized with sucrose
?. [Reagent setup]
Prepare assay buffer:168 mM Na2HPO4 and 10.6 mM KH2PO4,  , isotonized with KCl
?. [Sample preparation]
Euthanize fish in ice-cold water (Two brains should be pooled per to form one sample unit.
?. [Sample preparation]
Centrifuge samples on  tissue lysis buffer, at 1.000 x g for 5 min.
0.5 mL
?. [Sample preparation]
Reserve supernatants, which will be used for assays, and maintain  throughout the experiment.
on ice
?. [Incubation]
Mix protein samples (approximately 100 µg) with 460 µL of assay buffer and preincubated at   for  (5 min).
37 °C
?. [Incubation]
Start reaction by adding  kynuramine hydrobromide in a final volume of 700 µL.
?. [Incubation]
Incubate the final solution for  (30 min).
?. [Incubation]
Stop reaction with 300 µL  trichloroacetic acid.
Tricholoracetic acid is a strong acid that causes severe skin burns and eye damage, and may cause respiratory irritation. All solutions using TCA should be made using safety googles and nitrile rubber gloves, and vapors avoided by making preparations under a fume hood.
?. [Incubation]
Centrifuge reaction products at 16.000 x g for  (5 min) and 800 µL of the supernatants mixed with 1 mL NaOH (.
?. [Fluorescence measurement]
Transfer centrifuged, NaOH-buffered reaction products to quartz cuvettes
?. [Fluorescence measurement]
Transfer cuvettes to a spectrofluorophotometer
?. [Fluorescence measurement]
Measure fluorescence using excitation at 315 nm and emission at 380 nm.
?. [Fluorescence measurement]
Concentration should be estimated with a standard curve of 4-hydroxyquinoline (4-Quinolinol)
?. [Fluorescence measurement]
Calculate enzyme activity as nmol 4-OH quinoline/min/mg protein (determined by the Bradford or, preferably, Lowry assays).
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TITLE: Image quantification
AUTHORS: Chunmei  Chang

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

[STEPS]
?. GUV image quantification
?. Analyze GUV images using a custom script implemented in Python 3.6 (https://github.com/Hurley-Lab/GUVquantification/blob/main/GUVintensity-2channel.ipynb).
?. Bead image quantification
?. Define the outline of individual bead  based on the bright field channel.
?. Calculate the intensity threshold by the average intensities of pixels inside and outside of the bead.
?. Obtain the average intensity measurements of individual bead.
?. Calculate the averages and standard deviations among the measured values per each condition.
TITLE: UC Davis - Metabolomics: Sample preparation for Lipidomics
AUTHORS: Oliver Fiehn

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">This SOP describes sample extraction and sample preparation for lipid profiling by liquid chromatography / quadrupole time of flight mass spectrometry (LC-QTOF) or nanoelectrospray ion trap-FTICR MS.</div></div>

[STEPS]
?. 
?. Starting material: Plasma/serum: 30 µl sample volume or aliquot
?. Sample Preparation: Switch on bath to pre-cool at –20°C (±2°C validity temperature range)Extraction solvents    ♦ Purge both MeOH and MTBE for 5 min with nitrogen.    ♦ Store solvents in the –20°C freezer to pre-chillHomogenization and extraction    ♦ Thaw plasma on ice, and gently rotate or invert the blood samples for about 10s to obtain a homogenized sample.     ♦ Take out 60 µL and add 220 µL cold MeOH. Add 5 µL of QC mix as internal standard (see SOP “QC mix for LC-MS lipid analysis”).     ♦ Vortex each sample for 10s, keeping the rest on ice    ♦ Add 750 µL MTBE    ♦ Vortex for 10s    ♦ Shake for 6min at 4°C    ♦ Add 187.5 µL distilled water    ♦ Vortex for 20s     ♦ Centrifuge for 2 min @ 14000 rcf    ♦ Remove supernatant, splitting into two aliquots of 300 µL, keeping one at –20°C for backup    ♦ Dry samples to complete dryness in the speed vacuum concentration systemPreparation for analysis    ♦ Re-suspend dry samples in 70 µL MeOH containing CUDA (10 µM), degassed using the above method.     ♦Transfer 30 µL to two separate amber glass vial with micro-insert. Cap vials with Agilent blue top.    ♦ Use independent vials for positive and negative mode acquisitions.
?. Quality assurance   ♦ For each sequence of sample extractions, perform one blank negative control extraction by applying the total procedure (i.e. all materials and plastic ware) without biological sample.    ♦ Use one commercial plasma/serum pool sample per 10 authentic subject samples as control. If no suitable commercial blood sample is available, prepare a large pool sample during the thawing/mixing step by aliquoting 100 ul per 1 ml plasma sample, and aliquot such pool sample for 1 pool extract per 10 authentic subject samples.     ♦ Prepare at least six NIST plasma extracts in the same manner as positive controls
TITLE: Shipping beetles to the UF Forest Entomology Lab
AUTHORS: Jiri Hulcr

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to send specimens to the UF Forest Entomology Lab.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]