eamag/protocols-with-steps huggingface dataset:
TITLE: Biochemical Measures of Neuropathy - Glutathione Assay (GSH)
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/biwt4kcn.jpg" /></div></div>

[STEPS]
?. [Sample Preparation:]
Do not let samples set at 0º for long periods of time.Prior to dissection, perfuse tissue with PBS pH7.4 with 0.16 mg/ml heparin1. Label 6 sets of micro centrifuge tubes. (If only doing GSH only need 3 sets)2. Prepare 3 M HClO4 and freeze.3. Cut tissue into segments and weigh. (~10 mg)4. Keeping labeled tubes on dry ice, add 3 times the weight of 3 M HClO4.5. Sonicate on ~5.6. Add 300 μl of ddH²O for each 100 μl HClO4 (3.3 per μl) and vortex. (May include 1 mM EDTA in ddH²O)7. Centrifuged at 5000 x g for 10 min.8. Transfer supernatant to a new labeled tube. Discard protein precipitate.9. Immediately neutralize with 290 μl/ml of a mixture of 2 N KOH, 0.4 M imidazole base, and 0.4M KCl and vortex. (Leaving a solution at pH 7.)10. Centrifuge samples @ 5000 x g for 10 min @ 4º to precipitate insoluble KClO4
?. [Performing the assay:]
1. Set up Fluroskan layout and area. Read using 345nm excitation and 425nm emission.2. Prepare standards as follows: (0-1000uM)3. On a clear 96 well plate add 20 µl standards to each well in duplicate 4. Add 20 µl sample extract to each sample well in duplicate or triplicate. 5. Add 180 µl Tris-HCl buffer to each well. 6. Begin reaction by adding 2 µl methanol solution of o-phthaldialdehyde (OPD) (10 mg/ml methanol) 7. Place plate into Fluroskan holder and click START.8. From the drop down menu under sheet select curve fit. Choose the appropriate data to organize (usually Measure1),     then click OK. This calculates your standard curve.9. Save the curve fit sheet as an Excel file into your data folder. Use the naming convention GSXXXXXX.xls, where     XXXXXX is the date in yymmdd.xls.
TITLE: Culturing 3T3 Cells for Validating GEARBOCS Constructs
AUTHORS: Justin T Savage

[DESCRIPTION]
This protocol is for culturing Cas9 NIH/3T3 cells for the validation of GEARBOCS 2.0 and similar constructs for use in in vivo assays.

[STEPS]
1. Validation by indel sequencing in immortalized murine cell lines:
4. Seed Cas9 NIH/3T3 cells at 250k per well of a 6-well plate (if starting from frozen cells, you. will need to passage at least once prior to transfection. So thaw to 10cm dish, then passage to 6-well)
5. 24 hours after seeding, transfect Cas9 NIH/3T3 cells with GEARBOCS plasmid:
6. For each gene of interest, combine the following components in a 1.5 mL tube:
7. Mix by vortexing
8. Add 6 uL of Xtreme Gene
9. Pipette up and down rapidly. Do not touch pipette tip to the wall of the tube (Xtreme gene is sticky and extremely finicky. You want to minimize contact between XG and plastic surfaces)
10. Mix tube contents by gently flicking
11. Incubate 30 min at room temperature.
12. Add 200 uL of optimem/XG/DNA mixture per well in drop-wise swirl fashion.
13. Return cells to incubator for 48 hours
14. 48 hours after transfection, collect cells and isolate genomic DNA for Sanger sequencing:
15. Place 6-well plate on ice
16. Aspirate media and wash once with 1 mL of ice-cold PBS per well
17. Aspirate PBS wash and add fresh 1 mL cold PBS for collection
18. Use rubber scraper to collect cells into 1.5 mL tube and keep on ice
19. Use Qiagen DNeasy kit to isolate genomic DNA
6.1. 200 uL optimum
1 uL of GEARBOCS (at 1ug/uL)
2. Prepare HEK293 cell media
3. 1. DMEM 450ml
2. Fetal Bovine Serum 50ml
3. L-Glutamine, 2 mM, 5ml
4. Sodium Pyruvate, 1 mM, 5ml
5. Pen-Strep, 100 U/mL, 5ml
TITLE: No Title
AUTHORS: 

[BEFORE_START]
Make sure all the necessary programs and dependencies are installed on your PC or server and work correctly.


[GUIDELINES]
Test data is available on the European Nucleotide Archive (ENA) website
https://www.ebi.ac.uk/ena/data/view/PRJEB21005


[STEPS]
SECTION: Basecalling of raw nanopore data with Albacore software.
?. 
SECTION: Consensus calling of long 16S rRNA concatemerized reads with use of the INC-Seq algorithm.
?. 
SECTION: Correction of wrongly oriented reads and size filtration with a chopSeq algorithm.
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SECTION: Read binning and generation of OTUs with a nanoCLUST algorithm.
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SECTION: Download and install all the required software.
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a method for VSMC isolation from mouse aorta by an explant method adapted from Metz et al (2012).</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Aim: To evaluate the efficacy and tolerability of replacing tetracycline with high dose of amoxicillin in bismuth-based quadruple therapy for H.pylori eradication.</p>
<p> </p>
<p>Methods: This randomized, open label clinical trial study was performed on 228 patients with H.pylori infection and duodenal ulcer without the history of previous H.pylori treatment. Patients were randomly divided into two groups. Group one received metronidazole 500mg, bismuth subcitrate 240mg and amoxicillin 1000mg, all three times a day and omeprazole 20 mg twice a day (amoxicillin group)for14 days. The second group received metronidazole 500mg three times a day and bismuth subcitrate240mg and tetracycline HCL 500mg both four times a day and omeprazole 20 mg twice a day (tetracycline group), for 14 days. Evaluation for compliance and drugs side effects were done after two weeks. Eight weeks after treatment, H.pylori eradication rate was assessed by c13-urease breath test.</p>
<p> </p>
<p>Results: Two hundred twenty-eight patients were enrolled. There was no statistically significant demographic difference between two groups. Amoxicillin containing regimen achieved higher Eradication rate: 105 of 110 ,95.51% (95%CI 91.5% to 99.3%) and 88 of 105, 83.8% (95% CI 76.7% to 90.8%) by per-protocol analysis (p-value=0.005) and 92.9%(95%CI 88.1% to 97.6%) and 76.5% (95% CI 68.7% to 84.2%) by intention-to-treat (p-value=0.001) for amoxicillin and tetracycline groups, respectively. Adverse events were statistically more significant higher in Tetracycline group (65.2%)  amoxicillin group (43.4%) (P value = 0.001).</p>
<p> </p>
<p>Conclusion: Bismuth based quadruple therapy included high dose of amoxicillin and metronidazole has an acceptable H. pylori infection eradication rate with good tolerance in patients with duodenal ulcer. This can overcome treatment resistance in areas with high prevalence of metronidazole and clarithromycin resistance</p>

[STEPS]
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TITLE: PBS with 0.1% Sodium Azide
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the method for preparing PBS with 0.1% Sodium Azide for storage and/or processing of fixed tissue.</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>To MD analysis for one of our previous results of docking, for example to analyze the drug/target, almost 40 steps are required to finalize the MD results.</p>
<p> </p>
<p>The following protocol is according to our own experience in this project and was improved so that the errors were solved during the projects.</p>

[BEFORE_START]
<p style="text-align: justify;"><strong>Ubuntu Linux platform version 15.0.4 must be already installed.</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">Then because GROMACS needs CMake and FFTW tools developed for discrete Fourier transforms (DFTs), these were installed on Ubuntu too. FFTW was configured as:</p>
<p> </p>
<p><sub>$ --enable-threads --enable-sse --enable-float –enable-shared</sub></p>
<p> </p>
<p style="text-align: justify;">As installation of GROMACS is challenging and frequently got errors, it can be checked whether the GROMACS has been accurately installed by this command:</p>
<p> </p>
<p><sub>$ mdrun --version</sub></p>

[GUIDELINES]
<p style="text-align: justify;">This protocol was done using GROMACS v.5.0.4 under Ubuntu Linux platform version 15.0.4, with GROMOS  53A6 force field as provided by ATB version 2.2 (Lundborg and Lindahl, 2015).</p>
<p> </p>

[STEPS]
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TITLE: Open Field Test
AUTHORS: jillian.seiler

[DESCRIPTION]
The Open Field Test is a measure of rodent anxiety-like behavior. The rodent’s natural “anxious” tendency is to spend time in the outer borders of the chamber, close to the walls; this is called thigmotaxis. A rodent exhibiting decreased anxiety will spend more time in the center of the chamber, where it is unprotected by the walls. If only measuring anxiety-like behavior, measure time spent in the outside of the field and time spent in the center of the field (in this case, “center” refers to the 33cmx33cm square shape in the center of the field). You can also use the Open Field Test to look at locomotion (distance traveled and velocity), as well as exploratory behaviors (rearing).
This protocol is adapted from Tye, et al., 2013 (doi: 10.1038/nature11740) and was originally designed to be used with optogenetic stimulation. If you are performing an Open Field Test without stimulation, you can decrease the time to 10 minutes.

[STEPS]
SECTION: Procedure
1. Open Ethovision and apply necessary settings.
 

SECTION: Procedure
2. If not done already, swab Open Field with 70% Ethanol and let dry.
SECTION: Procedure
3. Grasp mouse lightly by the middle of the tail and place in center of the Open Field.
 

SECTION: Procedure
4. Press “Start” in Acquisition window of Ethovision.
SECTION: Procedure
5. When the animal has finished the test, gently remove it from the Open Field and return it to the
homecage.
 

SECTION: Procedure
6. Clean the Open Field with 70% Ethanol and paper towels, and continue with next animal.
SECTION: Analysis
7. Compare time in center and time in thigmo between groups, or, if using optogenetic stimulation, compare time in center and time in thigmo across 3 minute periods.
SECTION: Analysis
8. Assess freezing behavior (time spent frozen and instances of freezing).
TITLE: Senescence induction by DNA-damage in PCLS
AUTHORS: Delphine Beaulieu, Nayra Cardenes, Melanie Königshoff

[DESCRIPTION]
Precision-Cut Lung Slices (PCLS) are uniform, in size and thickness, tissue slices generated from human lungs that can be used for disease modeling, drug discovery and preclinical validation, while retaining the lung's cellular complexity and tissue architecture.
This protocol describes two different methods for the induction of cellular senescence by DNA damage in PCLS, and collection of samples for different analysis and measurements.

[BEFORE_START]
To start this procedure, you must have PCLS (Precision-Cut Lung Slices) ready (check protocol DOI dx.doi.org/10.17504/protocols.io.36wgq3xr5lk5/v1).

[GUIDELINES]
All steps involving human tissue must be conducted under BSL2 safety guidelines.

[STEPS]
SECTION: Day -1: PCLS acclimation to growth conditions
1. Add 1 mL of media per well in 24-well plates DMEM F-12 media ( ) + 1% FBS + 1% Penicillin-Streptomycin    + 0.3 µg/mL Amphotericin B   .

SECTION: Day -1: PCLS acclimation to growth conditions
2. Place one PCLS (1 cm diameter / 300 µm thick) per well with a brush.
SECTION: Day -1: PCLS acclimation to growth conditions
3. Incubate o/n (overnight) at 37 oC, 5% CO2.
SECTION: Treatment at Days 0, 2 and 4
4.2. Change media for each condition:

Control - Change media (above).
Bleomycin - 15 μg/ml Bleomycin in media above.
  
DMSO - 1:100,000 DMSO in media above.
  
Doxorubicin - 0.1 μM Doxorubicin in media above.
  
SECTION: Treatment at Days 0, 2 and 4
4. Treatment: change media with 1ml/PCLS, diluted in DMEM media + 0.1% FBS + 1% Pen/Strep + 1% Amphotericin B

SECTION: Treatment at Days 0, 2 and 4
4.1. Prior to media exchange, collect supernatant (s/n) from each condition in 1.5 mL tubes and, store at -80oC for future analysis.
SECTION: PCLS Harvesting for measurements
5.4. Day 0 and 6:
Snap freeze in liquid nitrogen: 4x PCLS per cryotube.
Store at -80oC for future analysis.
SECTION: PCLS Harvesting for measurements
5.3. Days 0 and 6:
Fix 2x PCLS with 1x fixative solution from β-galactosidase kit and stain for β-galactosidase as per manufacturer instructions.  

SECTION: PCLS Harvesting for measurements
5.2. Days 0 and 6:
Cell proliferation measurement: Use 2-3x PCLS to make 4x4 mm punches to perform WST-8 cell proliferation assay in a 96-well plate, following manufacturer's instructions.  

SECTION: PCLS Harvesting for measurements
5.1. Days 0, 2,4 6: 
Fix PCLS in 4% formaldehyde/PBS for 30 minutes (2x PCLS per condition).
  
Then wash twice with PBS and store at 4oC in PBS.
Use 1-2 slices for paraffin embedding, and posterior histological analysis.

SECTION: PCLS Harvesting for measurements
5. Collect PCLS at Day 0, 2, 4 and Day 6 for different baseline and final timepoint measurements.
TITLE: Kinase activity assays Src and CK2
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details about the Kinase activity assays of Src and CK2.

[STEPS]
1. To verify the activity of kinases SRC and CK2, add 45 µL of mixes containing either only kinase
assay buffer (25 millimolar (mM) Tris-HCl pH 7.4, 150 millimolar (mM) NaCl, 1 millimolar (mM) DTT, and 2 millimolar (mM) MgCl2), kinase buffer and substrate 
(0.5 mg/mL) or kinase buffer, substrate (0.5 mg/mL) and kinase (100 nanomolar (nM)) to individual wells of a Pierce white opaque 96-well plate (Thermo Scientific).
2. Substrate peptides used were RRRDDDSDDD 10-mer (PEP-CK2I-025, Biaffin) and Poly-(Glu,Tyr  4:1) (40217, BPS) for CK2 and Src kinases, respectively. For CK2, add a specific inhibitor Silmitasertib CX-4945 (S2248, Selleck- chem), where indicated, at a concentration of 1 micromolar (µM). 
3. Start the reactions by the addition of 5 µL ATP in kinase assay buffer, resulting in a final concentration of 100 micromolar (µM) ATP in each of the 50 µL reactions. 
4. After 60 min at Room temperature (RT) in darkness, add 50 µL of Kinase-Glo Max reagent (Promega) to each well, to reach a total volume of 100 µL. 
5. Allow the luciferase reactions to stabilize for 15 min before measuring luciferase activity at a Spark Multi-Mode Microplate Reader (TECAN). The luciferase activity correlates with ATP quantity, and thus, an inverse relationship between measured luminescence and kinase activity exists.
TITLE: Co-extraction of RNA and DNA from plant tissue
AUTHORS: Dominik Buchner

[DESCRIPTION]
This protocol describes how to co-extract RNA and DNA from plant tissue samples. Samples are homogenized and simultaneously lyzed by bead-beating. Cell debris is then caught with a pre-filter column, the DNA is then subsequently bound to a silica column, while the RNA passes the membrane. The RNA in the flow-through is then precipitated with 100% ethanol and bound to a second silica column. Both, DNA and RNA are washed with different wash buffers to remove remaining proteins and other contaminants and finally eluted in separate tubes. If the user is just interested in the RNA, the DNA spin-column can just be discarded.

[BEFORE_START]
Make sure all buffers are prepared before starting.

[GUIDELINES]
Follow general lab etiquette. Wear gloves to prevent contamination of samples. Clean the workspace before starting and after finishing with 80% EtOH.

[STEPS]
SECTION: Sample preparation and lysis
1. For each sample prepare one 2 mL screwcap tube pre-filled with approximately 400 mg of 2 mm zirconia beads and 0.1 mm glass beads.
SECTION: Sample preparation and lysis
2. Add up to 200 mg of plant tissue to the prepared tube.

 


SECTION: Sample preparation and lysis
3. Add 800 µL to the sample tube.

 


SECTION: Sample preparation and lysis
4. Immediately bead beat for 5 min at maximum speed.

 


SECTION: Lysate clearing and pre-filtering
5. 10 s 

SECTION: Lysate clearing and pre-filtering
6. Transfer 700 µL of the crude lysate to a pre-filter column. 

SECTION: Lysate clearing and pre-filtering
7. 10 min 

SECTION: DNA binding
8. Transfer 700 µL of the flowthrough from step 7 to a silica spin column to bind the DNA in the lysate. Keep the flow-through. Mark the spin column as the DNA column.

 


SECTION: RNA precipitation and binding
9. Add 350 µL to the flow-through from step 8 to adjust the binding conditions to bind RNA to the silica column.

SECTION: RNA precipitation and binding
10. Vortex the samples to mix the lysate with the ethanol. Do not centrifuge.
SECTION: RNA precipitation and binding
11. Load the mixture on a second spin column. Mark this column as the RNA spin column. 
11000 x g, 15 s  and discard the flow-through.

 


SECTION: Washing steps
12. Add 700 µL to the RNA spin column,11000 x g, 15 s  and discard the flow-through. 

SECTION: Washing steps
13. Add 500 µL  to the RNA spin column, add 500 µL to the DNA spin column, 11000 rpm, 15 s  and discard the flow-through.

SECTION: Washing steps
14. Add 500 µL  to the RNA spin column, add 500 µL  to the DNA spin column, 11000 rpm, 15 s  and discard the flow-through.

SECTION: Column drying and elution
15. 11.000 rpm, 1 min to dry the silica membrane of the spin columns. Transfer the spin column to a fresh 1.5 mL microcentrifuge tube.
SECTION: Column drying and elution
16. Add 100 µL directly to the silica membrane. Incubate the column for 3 min at Room temperature

SECTION: Column drying and elution
17. 11.000 rpm, 1 min , store the eluted RNA at -80 °C and the eluted DNA at -20 °C
TITLE: Agarose pads for microscopy
AUTHORS: Joao Vitor Molino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols describe the steps required for the preparation of agarose pads for live cell fluorescent microscopy. </div></div>

[STEPS]
?. [Before start]
Check all the necessary material:Glass slideCover slip Agarose (Agar should work as well, but probably with less imaging quality)Media or bufferChamber or well forming stick (ex: Frame-Seal™ in situ PCR and Hybridization Slide Chambers, 15 x 15 mm, 65 µl)Tube or flask for melting agarose1 mL plastic tips
?. [Melting agarose]
Melt the agarose in the desired concentration (were tested) using a microwave, with interval mixing. Using the desired liquid. (e.g. Media for keeping the cells alive)Keep the melted agarose in a plate heater at .
70 °C
?. [Glass slide preparation]
Cut a 1mL tips to increase the point diameter for easier liquid handling of the agarose.Separate a glass slide to use as cover during pad preparationStick the chamber forming material to a glass slide, maintaining the plastic barrier in the exposed side (Alternativaly you can follow the instructions at dx.doi.org/10.17504/protocols.io.kfrctm6 for a option without the chamber sticks)Add a excess of melted agarose to the well, and cover it with another glass slide for shaping the agarose padRemove the glass slide, by sliding it carefully to the sideStick the chamber forming material to a glass slide, maintaining the plastic barrier in the exposed side (Alternativaly you can follow the instructions at dx.doi.org/10.17504/protocols.io.kfrctm6 for a option without the chamber sticks)Add a excess of melted agarose to the well, and cover it with another glass slide for shaping the agarose padRemove the glass slide, by sliding it carefully to the side
?. [Sample addition]
Add the desired amount of sample to the agarose pad. (5-6  drops were tested | Be aware of cell concentration if the goal is to see individual cells)Wait the drops to dry to    (Specially important for cells with motility)Carefully remove the plastic barrier exposing the adhesive face of the well forming materialAdd a cover slip, sealing the well. (Avoids dehydration of the agarose pad, important for long microscopy experiments)
1 µl
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>We report a phylogeographic study of Chaetophractus villosus populations in Argentina. Control Region (CR) sequences (484 bp) were obtained for 76 C. villosus from 20 locations across the species whole distribution range. Seventeen new haplotypes were identified. The highest genetic variation and the earliest fossils were found in the Pampean Region, thus appearing as the most probable area of origin of the species. A general pattern of Contiguous Range Expansion (CRE) was revealed by Nested Clade Analysis (NCA) supported by mismatch analysis and Fu’s test. The Pampean Region would have been the preexpansion area, while Patagonia would have been the main dispersal route of contiguous expansion, possibly after the Pleistocenic glaciations.</p>
<p> </p>
<p> </p>

[GUIDELINES]
<p>Partial sequences of mtDNA Control Region (CR) are amplified using the universal primers Thr-L15926 (5´-CAATTCCCCGGTCTTGTAAACC-3´), located in the neighboring tRNA-pro gene and DL-H16340 (5´-CCTGAAGTAGGAACCAGATG-3´) (Vilá el al. 1999). </p>

[STEPS]
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