eamag/protocols-with-steps huggingface dataset:
TITLE: Imaging Mass Cytometry Data Acquisition
AUTHORS: Michelle Daniel, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SOP describes the image acquisition with an Imaging Mass Cytometer (IMC or</div><div class = "text-block">Hyperion).</div><div class = "text-block">Prior to this protocol the IMC needs to be fully tuned to specifications (see</div><div class = "text-block">“HuBMAP_Protocol_4_IMC_Tuning.pdf”). Additionally, every batch of samples stained</div><div class = "text-block">with the same antibody-mix requires the acquisition of a compensation slide for spill over</div><div class = "text-block">correction (see “HuBMAP_Protocol_3_Compensation_Slide_Preparation.pdf” &</div><div class = "text-block">“HuBMAP_Protocol_6_IMC_Compensation_Slide_Acquisition.pdf”).</div></div>

[STEPS]
?. [Set up the flat-bed scanner]
1. Attach the flat-bed scanner to a computer.
?. [Create overview images of the slides]
1. Use the flat-bed scanner with the custom sample holder and load up to 12 slides to be acquired.2. Scan the slides to .jpg at a resolution of 600 dpi and save the image.
The low resolution overview scanning of each slide can be performed on any computer.
?. [MCD file setup and Panorama creation]
1. Insert slide into the IMC.2. Under “Data acquisition” create a new Empty12AF file.3. Make sure that the autofocus pins in the software are not located above the hydrophobic markerpen. This may lead to the creation of a false focal plane image of the slide and result indecreased ablation efficiency.4. Save the mcd file in the data drive (“E”) as a new folder.5. Create panorama images of the tissue samples. Note: Panoramas should not be larger than10’000 x 10’000 pixels.
This requires a running, fully tuned IMC.
?. [ROI selection]
1. Locate the areas of interest identified in IHC staining in the panorama.2. Set up an ROI for the region(s) of interest with a defined area(s) (e.g. 1000 x 1000 pixels).3. In AirLab download the relevant panel (in options select “for CyTOF 2”). Save it to the folderwhere the mcd file is located.4. Select all ROIs and under “Template” import the panel downloaded from AirLab.Scroll to thebottom of the list and select the imported panel. In the panel selection pane, set the ablationenergy to 2.5 dB and the Ablation Frequency to 400 Hz.5. Double check that the right template was selected and that Ir191 and Ir193 are included.6. Hit select template.7. Set the DV Opt parameter to 4 h.8. Check the “Generate Text File” box.9. Hit start. (1 mm2 at 400 Hz should roughly take 1h).
Once the panel has been downloaded from AirLab and added to the first acquisition it isavailable in the IMC software. It will then not be necessary to repeat this step afterwards and it will alsonot be necessary to save the panel.conf to every single folder that is created for each slideID.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>MATERIALS</strong></p>
<p> </p>
<p>         1)      Overnight cultures of the bacterial recombinants to be screened</p>
<p>         2)      Agarose beads</p>
<p>         3)      Phenol:Chloroform (1:1)</p>
<p>         4)      Microfuge tubes</p>
<p> <br /><strong>Reference</strong><br /><br /></p>
<p>E. Beuken, C. Vink, and C.A. Bruggeman.  (1998.)  One-Step Procedure for Screening Recombinant Plasmids by Size.  <u>Biotechniques</u> <strong>24</strong>:  748-750</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Synthesis helps to facilitate the chemical production process. As a mature performance of synthesis technology, <a href="http://www.bocsci.com/carbohydrate-service.html" target="_blank">carbohydrate synthesis service</a> and <a href="http://www.bocsci.com/biosynthesis-of-chemicals.html" target="_blank">enzyme synthesis service</a> are easily accessed. Here is the protocol of impa (adenosine 5'-phosphorimidazolide) synthesis.</p>

[STEPS]
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TITLE: Purification mCherry-ATG13 IDR
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of mCherry-ATG13 IDR.

[STEPS]
SECTION: Purification
1. To purify mCherry-tagged ATG13 IDR, fuse the coding sequence for ATG13 (190-517aa) or ATG13 (230-517aa) to a N-terminal 6xHis-TEV-mCherry-tag through cloning into a pET-DUET1 vector (plasmids available from Addgene). 
SECTION: Purification
2. After the transformation of the pET-DUET1 vectors encoding the mCherry-tagged ATG13 IDR in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow the cells in 2x Tryptone Yeast extract (TY) medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C. 
SECTION: Purification
3. Once the cells reaches an OD600 of 0.8, induce the protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min at 18 °C. 

SECTION: Purification
4. Collect cells by centrifugation and resuspend in lysis buffer.  

Lysis buffer:

 
 
SECTION: Purification
5. Sonicate the cell lysates twice for 30 s. 
SECTION: Purification
6. Clear the lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 
SECTION: Purification
7. Filter the supernatant through an 0.45 µm filter and load onto a pre-equilibrated 5 mL His-Trap HP column (Cytiva). 
SECTION: Purification
8. After His-tagged proteins are bound to the column, wash the column with three column volumes of wash buffer.  

Wash buffer:

 
 
SECTION: Purification
9. Elute the proteins with a stepwise imidazole gradient (30, 75, 100, 150, 225, 300 mM). 
SECTION: Purification
10. Pool the fractions containing the 6xHis-TEV-mCherry-ATG13 IDR, concentrated using a 30 kDa cut-off Amicon filter (Merck Millipore). 
SECTION: Purification
11. Load the samples were ed onto a pre-equilibrated Superose 200 Increase 10/300 GL column (Cytiva). Elute the proteins with SEC buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM DTT).  

SEC Buffer:

 
 
SECTION: Purification
12. Analyze the fractions by SDS-PAGE and Coomassie staining. 
SECTION: Purification
13. Pool the fractions containing purified ATG13 IDR. 
SECTION: Purification
14. After concentrating the purified protein, aliquote the protein and snap-frozen in liquid nitrogen. 
SECTION: Purification
15. Store the proteins at -80 °C.
TITLE: High resolution Nano-DESI mass spectrometry imaging of proteomics of tissue
AUTHORS: yang1832 , Julia Laskin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">Acquire imaging mass spectrometry (IMS) datasets of proteins on human kidney tissue with ~12μm spatial resolution.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">Visualize the proteins distribution localizing to physiological regions within the human kidney by generating ion images of multiply charged protein peaks.</div></div>

[STEPS]
?. Scanned the tissue section with PathScan Enabler IV to visuslize the morphology in optical image.
?. Calibrate the instrument with Agilent tune mix.
?. Set up the appropriate method for proteins.
?. Place the slide on the slide holder and set up the primary capillary and secondary capillary and the solvent flow rate until there is liquid bridge between the two capillaries when land on the tissue surface. Direct the secondary capillary to the mass spectrometry inlet and clamp the capillary voltage on the syringe, adjust the positions of the capillaries untill there are stable and intensive signals in the spectrum.
?. Add protein standards into ACN:H2O:acetic acid=65:34:1 solvent as the extracting solvent. Add extracting solvent into the syringe.
?. Using the Labview software to train the shear force probe until it can appropriately recognize the sample surface from the air.
?. Place the shear force probe close to the liquid bridge so that it can lead the moving of the capillaries and enable the liquid bridge landing on the sample surface without scratching the tissue.
?. Set the start point and the end point of the scanning region.
?. Set up the worklist.
?. Start the acquisition.
TITLE: Embedding Rat Heart
AUTHORS: Shaina Robbins

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

[STEPS]
?. [Heart Removal]
Remove heart from rat immediately following sacrificeRinse heart in 1X PBS for  to flush blood from the chambers
?. [Embedding Heart]
Place heart (still beating) in solution of 1X PBS with 25% Optimal Cutting Temperature Compound (OCT) for  with gentle agitationPlace heart in solution of 1X PBS with 50% OCT for 3. Place heart in the embedding mold containing a layer of frozen 100% OCT at bottom. 4. Flash freeze the embedding mold containing heart in 100% OCT by suspending mold in either liquid nitrogen or methanol in dry ice5. Store heart in freezer wrapped in aluminum foil and plastic wrap until the sectioning process is started.
The heart should still be beating at this point, albeit weaker. This will allow for OCT infiltration into the chambers in order to mitigate any structural collapse while sectioning.
This layer of OCT is to help prevent the heart from sinking to the bottom of the mold exposing the tissue. All areas of the tissue sample should be covered with OCT to allow for optimal sectioning  capability.
Be careful to not allow any liquid nitrogen or methanol to splash into the embedding mold. This will make the OCT become a gooey texture and very difficult to section.
-80 °C
Allow heart to sit in  freezer for at least one day before beginning to section.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Since the 20th century, medical scientists represented by Paul Ehrlich, the father of chemotherapy, have been searching for such a "magic bullet" that distinguishes between pathogens and normal cells. Since the beginning of the new century, more and more people believe that antibody-drug conjugate (ADC) is the answer that has long been sought.</p>

[STEPS]
?.
TITLE: Automatic Multi-functional Integration Program (AMFIP) towards All-optical Mechano-electrophysiology Interrogation
AUTHORS: Qin  Luo, Justin  Zhang, Miao  Huang, Gaoming  Lin, Mai  Tanaka, Sharon  Lepler, Juan  Guan, Dietmar  Siemann, Xin  Tang

[DESCRIPTION]
Automatic operations of multi-functional and time-lapse live-cell imaging are necessary for the biomedical science community to study active, multi-faceted, and long-term biological phenomena. To achieve automatic control, most existing solutions often require the purchase of extra software programs and hardware that rely on the manufacturers’ own specifications. However, these software programs are usually non-user-programmable and unaffordable for many laboratories. To address this unmet need, we have developed a novel open-source software program, titled Automatic Multi-functional Integration Program (AMFIP), as a new Java-based and hardware-independent system that provides proven advantages over existing alternatives to the scientific community. Without extra hardware, AMFIP enables the functional synchronization of the μManager software platform, the Nikon NIS-Elements platform, and other 3rd party software to achieve automatic operations of most commercially available microscopy systems, including but not limited to those from Nikon. AMFIP provides a user-friendly and programmable graphical user interface (GUI), opening the door to expanding the customizability for myriad hardware and software systems according to user-specific experimental requirements and environments. To validate the intended purposes of developing AMFIP, we applied it to elucidate the question whether single cells, prior to their full spreading, can sense and respond to a soft solid substrate, and if so, how does the interaction depend on the cell spreading time and the stiffness of the substrate. Using a CRISPR/Cas9-engineered human epithelial Beas2B (B2B) cell line that expresses mNeonGreen2-tagged mechanosensitive Yes-associated protein (YAP), we show that single B2B cells develop distinct substrate-stiffness-dependent YAP expressions within 10 hours at most on the substrate, suggesting that cells are able to sense, distinguish, and respond to mechanical cues prior to the establishment of full cell spreading. In summary, AMFIP provides a reliable, open-source, and cost-free solution that has the validated long-term utility to satisfy the need of automatic imaging operations in the scientific community.

[STEPS]
TITLE: The Efficacy and Safety of a Radial Approach versus a Distal Radial Approach for Diagnostic Coronary Angiography and Percutaneous Coronary Intervention: A Systematic Review and Meta-analysis (protocol)
AUTHORS: Toshihide Izumida, Jun Watanabe, Ryo Yoshida, Kazuhiko Kotani

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">BACKGROUND </div><div class = "text-block">The traditional radial approach (RA) is recommended as the standard method for coronary angiography (CAG), while a distal radial approach (DRA) has recently been used for CAG.</div><div class = "text-block">AIM </div><div class = "text-block">The aim of this study will be to assess the efficacy and safety of DRA versus RA during CAG.</div><div class = "text-block">METHODS</div><div class = "text-block">The following databases will be searched through December 2020: MEDLINE, the Cochrane Central Register of Controlled Trials, EMBASE, the World Health Organization International Clinical Trials Platform Search Portal and ClinicalTrials.gov. Individual randomized-controlled trials for adult patients undergoing cardiac catheterization will be included. The primary outcomes will be the successful cannulation rate and the incidence of radial artery spasm (RAS) and radial artery occlusion (RAO). Study selection, data abstraction and quality assessment will be independently performed using the Grading of Recommendations, Assessment, Development, and Evaluation approach.</div></div>

[STEPS]
TITLE: Methanol-Chloroform-Water Precipitation
AUTHORS: Bryon Drown, Kelleher Research Group

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Methanol-chloroform-water (MCW) precipitation is a rapid method for removing interferring species from protein samples prior to LC-MS analysis. MCW precipitation can effectively remove both ionic and non-ionic detergents and salts. Precipitating less than 1 µg protein is challenging yet possible with careful pipetting.</div></div>

[STEPS]
?. Dilute protein sample to 150 µL with Optima-grade water.
?. Precipitate protein at interface
?. Pellet protein at interface
?. Wash pellet with methanol. (This step can be repeated if a high level of contaminating species is anticipated)
?. Redissolve protein in LC buffer
?. Add 600 µL Optima-grade methanol and mix well.
?. Add 150 µL HPLC-grade chloroform and mix well.
?. Centrifuge and discard top layer while being careful to not disturb interface between layers. Use a gel-loading tip to remove last bit.
Centrifuge: 20000 34, 10 min, 4 10
?. Add 600 µL and homogenize the solvent, being careful not to break up the precipitated protein.
?. Centrifuge   and discard supernatant, being careful not to disturb pellet.
Centrifuge: 20000 34, 4 10
?. Add 600 µL methanol and mix by inverting tube.
?. Centrifuge  and discard supernatant, being careful not to disturb pellet.
Centrifuge: 20000 34, 4 10
?. Evaporate excess methanol by laying tube on its side for 5 min at room temperature.
?. Redissolve precipitated protein in 30 µL Buffer A. Incubate 10 min on ice. If protein is not completely dissolved, sample can be briefly sonicated with a bath sonicator.
?. Centrifuge   and transfer supernatant to LC sample vial.
Centrifuge: 20000 34, 4 10
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes a procedure how primary metabolites and proteins can be extracted from <em>Deinococcus radiodurans</em> simultaneously. Polar metabolites are purified and derivatized for GC-MS (TOF) analysis. Proteins are tryptically digested in-gel and peptides are prepared for bottom-up proteomics by LC-MS (orbitrap).</p>

[STEPS]
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TITLE: ONT DNA Barcoding Fungal Amplicons w/ MinION &amp; Flongle
AUTHORS: Stephen        Douglas Russell

[DESCRIPTION]
This collection of protocols outline a working methodology to DNA barcode fungal specimens. This process will work for dried tissue, fresh tissue, or with DNA template that has already gone through an alternative extraction protocol. It takes you from computer/software setup through DNA extraction, amplification, library preparation, sequencing, and primary data analysis.

This particular workflow is designed for use with Flongle 9.4.1 flowcells and V12 Ligation chemistry.


[STEPS]
TITLE: Chloramphenicol 25 mg/mL Stock Solution
AUTHORS: Bailey  Clark

[DESCRIPTION]
The abstract will be added later.

[STEPS]
SECTION: Preparing the Stock Solution
1. Weigh 25 mg (0.025 g ) of Chloramphenicol.

SECTION: Preparing the Stock Solution
2. Add the chloramphenicol to a 1.5 mL tube. 
SECTION: Preparing the Stock Solution
3. Add 100% ethanol to the 1mL level on the tube.
SECTION: Preparing the Stock Solution
4. Vortex the tube for 5 seconds, repeat 3 times. 
SECTION: Preparing the Stock Solution
5. Store the tube in the -20°C freezer.
TITLE: Endometrium dissociation with trypsin 
AUTHORS: Regina Hoo, Roser Vento-Tormo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is for enrichment of epithelial glands on endometrium; following the step from "Endometrium dissociation with collagenase".</div></div>

[STEPS]
?. Following step 10 from "Endometrium dissociation with collagenase" protocol, where pieces of tissue are retained on the cell strainer. Collect pieces retained on the filter by inverting the filter into a new 50 mL tub and adding 45 ml of PBS with a 1 mL pipette.
?. Centrifuge at 450 g for  together with collagenased sample. Discard supernatants.
?. Resuspend the pieces with   Trypsin/EDTA 0.25% and incubate for  at while shaking.
10 ml
37 °C
?. Add of RPMI with 10% FBS.
20 ml
?. Filter material slowly and carefully (very dens liquid) through a 100 μM strainer. Discard retained tissue.
?. Centrifuge at 500 rcf for   at  . Discard supernatant. Same step as 14 in  "Endometrium dissociation with collagenase" protocol
4 °C
?. {optional} Resuspend sample with   of RLB mix and incubate for *RLB preparation: Dilute 10x RLB stock with water After RBC lysis, add of PBS and centrifuge at 500 rcf for
5 ml
10 ml
?. Resuspend cells in   of PBS in an Eppendorf.
1 ml
?. Count live cells using tryphan blue and haematocytometer.
?. Centrifuge at 500 rcf for   at . Discard supernatant.
4 °C
?. Resuspend with  of  PBS (final volume depending on cell number)
250 µl
?. Prepare Trypsin-EDTA-DNaseI mix:  ABCD1ProductStockFinal volume (10 ml)Concentration2Trypsin-EDTA (0.25%) phenol red9.9 ml3DNaseI10mg/ml100 ul0.1 mg/ml
ABCD1ProductStockFinal volume (10 ml)Concentration2Trypsin-EDTA (0.25%) phenol red9.9 ml3DNaseI10mg/ml100 ul0.1 mg/ml
?. Filter cells trough a cell strainer snap cap tube.
?. {Optional} Resuspend cells in freezing medium to a concentration of 1x 107  cells and aliquot into cryogenic storage vials.
TITLE: Immunoprecipitation (IP) and Mass Spectrometry
AUTHORS: Yingchao Xue, Xiping Zhan, Shisheng Sun, Senthilkumar S. Karuppagounder, Shuli Xia, Valina L Dawson, Ted M Dawson, John Laterra, Jianmin Zhang, Mingyao Ying

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol explains Immunoprecipitation (IP) and Mass Spectrometry for lines ND1014, N1, and ND27760 from </span><span style = "font-style:italic;">Synthetic mRNAs Drive Highly Efficient iPS Cell Differentiation to Dopaminergic Neurons</span><span>. </span></div></div>

[STEPS]
?. [Immunoprecipitation (IP)]
Twenty-four hours after mRNA transfection, extract nuclear proteins of iPSCs using the NE-PER Nuclear and Cytoplasmic Extraction Kit.
?. [Immunoprecipitation (IP)]
Perform FLAG IP using anti-FLAG M2 antibody and Protein G Dynabeads following the manufacturer’s protocol.
?. [Immunoprecipitation (IP)]
Elute binding proteins by elution buffer (, ).
[urea]
[NaHCO3 in water]
?. [Mass Spectrometry (MS)]
Denature protein samples in /.
[urea]
[NH4HCO3 buffer]
?. [Mass Spectrometry (MS)]
Reduce protein samples by  at  for .
[TCEP]
37 °C
?. [Mass Spectrometry (MS)]
Alkylate protein samples by  at  in the dark for .
[iodoacetamide]
37 Room temperature
?. [Mass Spectrometry (MS)]
Dilute samples fivefold with deionized water.
?. [Mass Spectrometry (MS)]
Incubate at  with shaking.
37 °C
?. [Mass Spectrometry (MS)]
Centrifuge at  to remove any particulate matter.
Centrifuge: 13000 34, 10 min
?. [Mass Spectrometry (MS)]
Purify by a C18 solid-phase extraction.
?. [Mass Spectrometry (MS)]
Elute peptides from the C18 column in 60% ACN/0.1% TFA.
?. [Mass Spectrometry (MS)]
Measure peptide concentrations using a NanoDrop microvolume spectrophotometer.
?. [Mass Spectrometry (MS)]
Resuspend in 0.2% FA solution.
?. [IP and MS Analysis]
Search raw data against UniProt human protein database by Proteome Discoverer with the following parameters: two missed cleavages allowed for trypsin digestioncarbamidomethyl (C) set as a fixed modificationoxidation (M) and acetyl (protein N-terminal) set as variable modifications
?. [IP and MS Analysis]
Normalize the intensity of each identified proteins to the intensity of their A-WT or A-SA proteins, respectively, and further calculate the A-SA/A-WT ratio of normalized intensity.
?. [Mass Spectrometry (MS)]
Add sequencing grade trypsin (50:1, w/w) to the samples.
?. [Mass Spectrometry (MS)]
Inject 1 μg in 6 μl solution for each LC–MS/MS analysis using Velos Orbitrap mass spectrometer with a 120-minute online LC separation.
TITLE: Olfactory mucosa sampling and processing 
AUTHORS: maria.xylaki

[DESCRIPTION]
This protocol describes the olfactory mucosa sampling and processing for RT-QuIC assays and was adapted from A. Stefani et al. (doi:10.1093/brain/awab005).

[STEPS]
SECTION: Olfactory mucosa sampling
1. Two olfactory mucosa samples are collected per person, one nasal swabbing from each nostril.
A sterile, disposable swab is inserted in the nasal cavity, (similar to a covid test but more towards the top) to reach the olfactory and not the epithelial mucosa. 
The swab is gently rolled on the mucosal surface, removed, and the tip is cut and placed in a 15-ml conical centrifuge tube with 0.9% NaCl solution.

SECTION: Olfactory mucosa processing
3. Swab is removed from the tube and the cell suspension is centrifuged at 2000 x g for 20 mins at 4 °C.
SECTION: Olfactory mucosa processing
4. Supernatant is removed and the pellet is directly processed or frozen at -80 °C for processing later.
SECTION: Olfactory mucosa processing
5. An inoculation loop is used to collect equal amounts of pellet (∼ 2 μl) which are then dipped into a tube containing 120 μl PBS.
SECTION: Olfactory mucosa processing
6. Pellets are disolved by sonicating the tube in a bath sonicator at 120 W for at least 1 min.

SECTION: Olfactory mucosa processing
7. Samples can be directly used for RT-QuIC assay.
SECTION: Olfactory mucosa processing
2. Olfactory mucosa cells are extracted from the swab by vortexing the tube for 1 min at room temperature.