eamag/protocols-with-steps huggingface dataset:
TITLE: U Mass - STZ-induced type 1 diabetes model
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">Streptozotocin can selectively destroy the pancreatic β-cells with rapid and irreversible necrosis and can be used to generate a chronic model of hyperglycemia and type 1 diabetes.</div></div>

[STEPS]
?. Administer an intraperitoneal injection of streptozotocin (50 mg/kg body weight) daily for 5 days.
?. Monitor glucose level for onset of hyperglycemia.
TITLE: Immunofluorescence staining of PFA or fresh frozen mouse brain section
AUTHORS: Daniel Manrique-Castano

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is suitable for immunofluorescence staining of PFA fixed or Fresh Frozen mouse brain sections.</div></div>

[STEPS]
?. [Rehydration and permeabilization]
If fixed sections are kept in  , rehydrate them before starting the staining procedure by incubating themin PBS for  .
-80 °C
For fresh frozen sections kept at   , incubate with   PFA during   followed by 3 washes in PBS,   each.
?. [Rehydration and permeabilization]
To permeabilize and block for unspecific staining, decant the PBS and incubate the section with Blocking-Permeabilization buffer for  at  in al humid chamber/box to prevent sections from drying out.
0 Room temperature
If required, encircle brain sections using a hydrophobic pen previous incubation to prevent buffer leakage. Ensure that all sections are well covered with buffer.
?. [Antibody incubation]
When permeabilization is achieved, decant the buffer and incubate the sections in primary antibody buffer containing diluted primary antibodies  at .
4 °C
?. [Antibody incubation]
When incubation time is finished, wash the sections 3 times with PBS,  each.
?. [Antibody incubation]
Incubate the cells in secondary antibody buffer containing diluted secondary antibodies  at
0 Room temperature
?. [Antibody incubation]
Subsequently,  wash the cells 3 times with PBS,  each.
?. [Antibody incubation]
To mount the sections, Remove as much of the remaining buffer as possible from the crystals, and with a pipette tip drop 1-2 small drops of  . Subsequently, cover the sections with appropriate glass coverslips.
?. [Antibody incubation]
Let section air dry in the dark before imaging.
TITLE: Custom Peptide Design
AUTHORS: Cathy  Miller

[DESCRIPTION]
Interested in the use of biologically active proteins and peptides as potential therapeutic agents has grown dramatically in recent years. Although many meaningful studies can be performed, it is important to consider several factors such as the length of sequence, amino acid residues and the sequence of peptides, all of which will influence whether correct assembly and purification are feasible. If you would like Creative Peptides to help you in designing your peptide, our technical support department can assist you.

[STEPS]
TITLE: Stable transfection of unicellular relative of animals,  Corallochytrium limacisporum, using Lonza Nucleofector
AUTHORS: Aleksandra Kozyczkowska, Sebastian Najle, Eduard Ocaña-Pallarès, Cristina Aresté, Iñaki Ruiz-Trillo, Elena Casacuberta

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol describes the transfection of </span><span style = "font-style:italic;">Corallochytrium limacisporum</span><span>, one of the unicellular relatives of animals. The transfection is performed using Lonza Nucleofector System X and reporter cassettes that contain endogenous regulatory elements of </span><span style = "font-style:italic;">C. limacisporum.</span><span style = "font-style:italic;"> </span></div></div>

[STEPS]
?. [Cell Culture preparation ]
Inoculate 100 μl of C. limacisporum culture in 5ml of Marine Broth in 25cm2 flask. Keep at 23°C air incubator for two days.
?. [Cell count]
Scrape gently and count cells to reach 1.5E6 cells / condition using hemocytometerIf there are more conditions, it is recommended to pull the cells together to get more visible pellet
?. [Medium removal]
Spin down the cells at 1500xg for 5'Discard the supernatant.
?. [Washing step]
Wash the pellet with an ice-cold 1XPBS (100 μl / condition)Spin down and discard the supernatant.
?. Transfer the mix (cells + DNA plasmid) into a strip (from the manufacture kit)Do it carefully & without creating bubblesFinal volume: 20-25 μl
?. [Transfection of plasmid DNA into C. limacisporum ]
Insert into Lonza nucelofection system and apply code: EN - 138
?. [Recovery step]
After transfection, immediately add 80 μl of Marine Broth medium (MB) to a wellMix up and down
?. [Check-out for results]
Check for positive cells 24 - 48h post-transfection using fluorescent microscopy
?. [Enrichment & selection of transfected cells]
Replace 500 μl of medium with fresh MB that contains 300 μg/ml of puromycinIncrease of mCherry-positive cells can be observed over time.
?. Add DNA mix to the cell pellet
?. [Prepare DNA mix]
Mix  DNA plasmids: 10 μg of an reporter plasmid  + 20 μg carrier DNA (pUC19) It is recommended that plasmid is highly concentrated  (2-5 μg/ul) to avoid final buffer dilution with the DNA solutionOur plasmid contains mCherry coding sequence & resistance to puromycin gene (pac) under constitutive promoter: actin (Addgene: #104446) or tubulin (Addgene#129560)Note: 10 μg of reporter plasmids guarantees the highest efficiency, however a successful transfection can be also obtained with 5 and 1μg of the plasmid
?. [Prepare DNA mix]
Transfer DNA mix to 20 μl of ice-cold P3 buffer It is recommended not to keep the cells in P3 buffer for too long
?. [Recovery step]
Transfer to 12-well plate (NUNC) that contains 1 ml of growth medium (MB) and incubate overnight
TITLE: Protocol: Neutrophil/lymphocyte ratio and overall survival in patients with breast cancer: a cohort study in a Latin-American hospital
AUTHORS: Martha Sofia Cervera-Ocaña, Jhony A. De La Cruz-Vargas, Dante Manuel Quiñones-Laveriano, Nataly Briyit Huamán Córdova

[DESCRIPTION]
PURPOSE OF STUDY
The primary objective of this study will be to determine the association between NLR and OS in patients with breast cancer in the gynecology department of Hospital Nacional Hipólito Unanue (HNHU), Lima, Peru between 2012 and 2014. 

[STEPS]
SECTION: Neutrophil/lymphocyte ratio and overall survival in patients with breast cancer: a cohort study in a Latin-American hospital
1.
TITLE: MRI_preprocessing_protocol
AUTHORS: Miguel López-Aguirre, José A. Pineda-Pardo, Jose Obeso

[DESCRIPTION]
Preprocessing protocol for structural, DWI and T2* MRI acquisitions

[STEPS]
SECTION: Structural T1w (MP-RAGE acquisition)
1. Data transformation from DICOM to NIFTI standard (software: dcm2niix)

SECTION: Structural T1w (MP-RAGE acquisition)
2. Field of view optimization (command: fslreorient2std; software: FSL)
SECTION: Structural T1w (MP-RAGE acquisition)
3. Bias field correction (command: N4BiasFieldCorrection; software: ANTs)
SECTION: Structural T1w (MP-RAGE acquisition)
4. Non-brain tissue removal (command: bet; software: FSL)
SECTION: Structural T1w (MP-RAGE acquisition)
7. Diffeomorphic registration to MNI152 2009c sym tempate (command: antsRegistrationSyN.sh; software: ANTs)
SECTION: Structural T1w (MP-RAGE acquisition)
5. Deep-brain nuclei segmentation (command: run_first_all; software: FSL)
SECTION: Structural T1w (MP-RAGE acquisition)
6. CSF, GM and WM tissue segmentation (command: fast; software: FSL)
SECTION: T2* (multi-echo gradient echo acquisition)
8. Data transformation from DICOM to NIFTI standard (software: dcm2niix)

SECTION: T2* (multi-echo gradient echo acquisition)
9. Affine registration to structural T1w using the first volume as reference (command: antsRegistration; software ANTs)
SECTION: Single-shell DWI (single-shot 2D spin-echo, b=1000)
10. Data transformation from DICOM to NIFTI standard (software: dcm2niix)
SECTION: Single-shell DWI (single-shot 2D spin-echo, b=1000)
11. B0 pair isolation (AP and PA)
SECTION: Single-shell DWI (single-shot 2D spin-echo, b=1000)
12. Image denoising using the overcomplete local PCA appoach (MATLAB)
SECTION: Single-shell DWI (single-shot 2D spin-echo, b=1000)
14. Eddy current-induced distortion corrections (command: eddy; software: FSL)
SECTION: Single-shell DWI (single-shot 2D spin-echo, b=1000)
13. Geometric distortions and magnetic field inhomogeneities corrections (command: topup; software: FSL)
SECTION: Single-shell DWI (single-shot 2D spin-echo, b=1000)
16. Affine registration to structural T1w using the first volume (B0) as reference (command: antsRegistration; software ANTs)
SECTION: Single-shell DWI (single-shot 2D spin-echo, b=1000)
15. Bias field correction (command: N4BiasFieldCorrection; software: ANTs)
TITLE: Production: 10x TBE Buffer Powder Sachets
AUTHORS: Jenny  Molloy, Nadine    Mowoh, Stephane  Fadanka

[DESCRIPTION]
Desired concentrations of Components in the 10x buffer
Boric acid-0.89M
Tris-0.89M
Na2EDTA-0.02M


Concentration of 1x TBE buffer
Tris-Borate-0.089M
Na2EDTA-0.002M

[BEFORE_START]
Ensure all powders are dry and free from moisture to enable the resulting buffer to stay longer.

[STEPS]
1. Weigh about 35 g of Boric acid into a weighing boat and place the weighing boat in a vacuum tupperware half-filled with silica gel beads.



2. Close the tupperware and use the vacuum pump to create a vacuum until you are not able to hand pump any more air out or the pump shuts off.
 

3. Allow it to stay overnight at room temperature 
 

4. Weigh out 27.515 g of  from the pre-dried powder into a 500 mL beaker.

5. Weigh 3.72 g of  and add into the beaker

6. Weigh 53.905 g of and add to the content of the beaker.

7. Use a spatula to stir so as to have an even and a uniform mixture of all three powders.

 

8. Transfer the powder mix into a zip lock antistatic bag, seal carefully and label appropriately as powder to make up 0.5 L of 10x TBE.

9. The powder is stable for up to 6 months.
TITLE: Western blot for tissue extract
AUTHORS: María Sanchiz Calvo, eduard.bentea, Veerle Baekelandt

[DESCRIPTION]
Protocol for the detection of proteins by Western blot from tissue extract 

[BEFORE_START]
Perform protein extraction from snap-frozen brain tissue:

- weigh tissue
- add RIPA buffer (see Materials) : 10 X of the weight (40 mg = 400 µL)
- homogenize samples using sample homogenizer
- sonicate samples at 4 degrees C, 3 times 15 seconds (keep the samples on ice between each sonication)
- centrifuge samples at 6000 g for 10 minutes at 4 degrees C
- collect supernatant and measure protein concentration
- aliquot protein extracts and store at -20 degrees 

[STEPS]
SECTION: Day 1
1. Prepare sample for western blot
15 µg of protein in 12 µL 
Add 4 µL 
Boil at 98 °C for 10 min      

SECTION: Day 1
2. Load samples, together with 7 µL   of mass marker (PageRuler‱ Plus Prestained Protein Ladder) on a on 4–15% Criterion‱ Tris-HCl Protein Gel.

SECTION: Day 1
3. Run the gel for 10 min at 80V

SECTION: Day 1
4. Run the gel for 45 min at 150V

SECTION: Day 1
5. Transfer the proteins on PVDF membrane (Trans-Blot‱ TurboTM PVDF Membrane) using Trans-Blot Turbo Transfer System (Bio-Rad), using the pre-programmed protocol STANDARD SD: 30 min   , up to 1.0 A, 25 V.

SECTION: Day 1
6. Block membranes for 60 min    using 5% milk dissolved in PBS-T 0,1% at RT Room temperature 

SECTION: Day 1
7. Incubate membranes    in primary antibody in 5% milk PBS-T 0,1% at 4 °C 

SECTION: Day 2
8. Wash membranes with PBS-T 0,1% for 10 minutes, 3 times at TRoom temperature 
Wash PBS-T 10 min 
Wash PBS-T 10 min
Wash PBS-T 10 min
9. Incubate membranes for 60 min horseradish peroxidase-conjugated secondary
antibody (Dako)  diluted 1/10000 in 5% milk PBS-T 0,1% at Room temperature 

10. Wash membranes with PBS-T 0,1% for 10 minutes, 3 times at TRoom temperature 
Wash PBS-T 10 min 
Wash PBS-T 10 min
Wash PBS-T 10 min
11. Develop membranes using ECL Prime (GE Healthcare)
TITLE: Thigmotaxis_Detection_2018
AUTHORS: Akinori Higaki, Masaki Mogi, Jitsuo Higaki, Masatsugu Horiuchi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We share the minimal dataset for our study "Recognition for Early Stage Thigmotaxis in Morris Water Maze Test with Convolutional Neural Network" here. This study is currently submitted for PLoS one.</div><div class = "text-block">any_cropper.txt : Python script for converting bmp images to resized grayscale data.</div><div class = "text-block">bin_recog.txt : Python script for distinguishing thigmotaxis from other trajectories.</div><div class = "text-block">six_recog.txt : Python script for classifying 6 specific trajectories.</div><div class = "text-block">any_recog_data_0309.xlsx : Data used for analysis of mice characteristics.</div></div>

[STEPS]
TITLE: HuBMAP UF TMC - 10x Genomics scRNAseq Modality Overview 
AUTHORS: Marda Jorgensen, Maigan Brusko

[STEPS]
?. Specimen/Organ Prep:Spleen: dx.doi.org/10.17504/protocols.io.bc3kiykwLymph Node: dx.doi.org/10.17504/protocols.io.bbgnijveThymus: dx.doi.org/10.17504/protocols.io.bbgmiju6
?. Single Cell Isolation/Storage: dx.doi.org/10.17504/protocols.io.bd9vi966
?. 10x Library Construction and Sequencing: dx.doi.org/10.17504/protocols.io.bffvjjn6
?. Data Analysis and Quality Control Check
?. ​Register completed 10x sample information in the HuBMAP UUID generator at https://uuid.hubmapconsortium.org.
?. Prepare and upload data to the HuBMAP HIVE  Globus using hipergator.
?. Optional (recommended) step: FACS isolation of live single cells for scRNASeq: dx.doi.org/10.17504/protocols.io.bfqzjmx6
TITLE:  CompetentCell
AUTHORS: Ana Belem García González, Georgina Diego, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz

[DESCRIPTION]
A protocol for making chemically competent cells is shown.

[STEPS]
SECTION: Competent cells protocol with CaCl2
1. Inoculate 100 µL of E. coli in10 mL of antibiotic-free starter LB culture medium. Incubate during  at 37 °C and    

SECTION: Competent cells protocol with CaCl2
2. Heat the LB media in the water bath at37 °C

SECTION: Competent cells protocol with CaCl2
3. Inoculate 40 mL of LB media with 1 mL of cell culture from step 1. 

SECTION: Competent cells protocol with CaCl2
4. Incubate at 37 °Cwith constant agitation (  during 30 min).

SECTION: Competent cells protocol with CaCl2
5. Measure de optical density at 600 nm every 30 min until an OD600 of 0.4-0.6 is reached.

SECTION: Competent cells protocol with CaCl2
6. Chill for20 min on ice 

SECTION: Competent cells protocol with CaCl2
7. Centrifuge during 15 min at 4 °Cand 3000 rpm

SECTION: Competent cells protocol with CaCl2
8. Discard the supernatant, add 20 mL of   solution 50 mM and resuspend carefully.

SECTION: Competent cells protocol with CaCl2
9. Chill for 20 min on ice

SECTION: Competent cells protocol with CaCl2
10. Centrifuge during 15 min at 4 °Cand 3000 rpm

SECTION: Competent cells protocol with CaCl2
11. Discard the supernatant, add 5 mL of CaCl2 solution 100 mM and resuspend carefully. Chillon ice for 20 min

SECTION: Competent cells protocol with CaCl2
12. Preserve at 4 °C
TITLE: cosy_metab.nan
AUTHORS: NAN KB, John Glushka, Mario Uchimiya, Saraa Al Jawad, Christopher Esselman, Leandro I Ponce, Laura Morris, Arthur Edison

[DESCRIPTION]
This is a protocol for running the Bruker pulse program "cosygpprqf" for metabolomics samples.

[BEFORE_START]
This protocol assumes:
Your sample is loaded, locked, tuned, and shimmed in the magnet
The calibrated 90° pulse value for proton (i.e., P1) for the sample has been collected

[GUIDELINES]
This protocol intends to provide concise instructions to carry out the experiment. For more comprehensive information, see Bruker's documentation "Basic NMR Experiments" by clicking ? →  Manuals (docs) on the menu bar on TopSpin. See also "Pulse Program Catalogue. 1D/2D" for the details about the pulse program used in this protocol.

[STEPS]
SECTION: Create a new dataset
1. 
SECTION: Create noesypr1d experiment file
0.23. When Lock, Tune, Shim are complete proceed to the specific protocol for your desired pulseprogram(s).
SECTION: Create a new dataset
1.1. On the menu bar on TopSpin, click on
Start → Create Dataset
 

 

SECTION: Create a new dataset
1.2. Enter
NAME: Name of a set of datasets (e.g., human_serum_study1). Use a single string.
EXPNO: Dataset number. Use a positive integer.

Select
Directory: Your directory.
 

 

SECTION: Create a new dataset
1.3. Select
Read parameterset

Click the button
Select
SECTION: Create a new dataset
1.4. A new window opens. On the right top bar, select
Source = /opt/NAN_METAB/par
 
In the list, select the one you want to use:

For serum and plasma samples:
COSY_br600_serum.par: Parameter set optimized for serum samples.

For urine samples:
COSY_br600_urine.par: Parameter set optimized for urine samples.
COSY_NUS_br600_urine.par: Parameter set using an acquisition mode "non-uniform sampling (NUS)". Higher resolution on the indirect dimension

 

Click
OK
SECTION: Create a new dataset
1.5. Click
OK
SECTION: Acquire a spectrum
2. Go to the "USE DEFAULT" tab below to proceed with the default optimized parameters.
3. 
3.1. Load the calibrated P1 using the following command in the command line.

 
(e.g., getprosol 1H 10.01 -7.45)

 

3. 
3.1. Select the AcquPars tab to show a parameter set.
 


 

3.2. Click the icon
_∏_
to see a complete parameter set.

 

3.3. Change parameters as needed.

Typically, the main parameter to adjust is NS:
 

3.4. Other potential parameters to adjust:
 

3.5. Other potential parameter for the NUS parameter sets:
 

3.6. Acquire a spectrum. If more optimization is needed, go to step 3.3.

 

3.2. Click on
Acquire → Gain
in the menu bar to automatically set the receiver gain.
 

 

3.3. Click
Go
in the menu bar to acquire a spectrum.
 

3.4. After the run, click on
Process → Proc. Spectrum
in the menu bar to execute an automated processing macro.
 

3.5. If you want to modify parameters to improve your spectrum, go to step 2 and move to the step case "MODIFY PAR".
TITLE: Ultra-high field, multi-echo MRI acquisition for resting-state functional connectivity
AUTHORS: Elizabeth Rizor, Dr. Scott Grafton

[DESCRIPTION]
This is an MRI acquisition sequence used to examine resting-state functional connectivity of open and closed loop motor circuits in healthy controls and those with Parkinson’s Disease. Scans include a Localizer, anatomical T1 MPRAGE, CMRR spin-echo AP and PA echo-planar imaging (EPI) images for field map generation, and a modified CMRR multi-echo EPI sequence for resting-state imaging. All participants complete a 30-minute MRI protocol at the University of Southern California’s Center for Image Acquisition, where they are scanned with a Siemens 7T Terra scanner and a research-only ​​8-channel transmit 32-channel receive Nova 8Tx/32Rx head coil (Nova Medical Inc.) 

[STEPS]
TITLE: Water Sampling and Plating Generations (Antimicrobial Group)
AUTHORS: Melissa B Duhaime, Aubrey Reed, Nicole Sunshine, Renae Lyons

[DESCRIPTION]
Protocol developed by the Antimicrobial Group class project. The goal was to isolate microbes from freshwater lakes that produce antimicrobial compounds in order to study antibiotic production in nature and environmental reservoirs of antibiotic resistance.

Protocol for class project in Microbes in the Wild: Advanced Environmental Microbiology Lab (EEB 447) course at University of Michigan Biological Station and on campus. 


[STEPS]
1. Collect whole water at six sites using a Van Dorn water collector for deep (~122.5-123.7 m) samples  and 2 L containers for surface samples (~15 cm below the surface). 
2. Set site names and classifications
 
3. Prefilter whole water using 20μm mesh. 
4. Set up peristaltic pump with 3 µm filter
5. Filter approximately 500mL of sample water.

6. Collect the filter in an eppendorf with 1mL of PBS as the particle-associated large fragment
3. Collect the <3μm filtered water as the free-living (FL) smaller fraction.
4. For the particle-associated (PA) fraction, vortex tube, then partially remove filter with an edge under the closed cap
5. Centrifuge at 1500 RPM for 1 minute

6. Fully remove filter and resuspend pellet

7. Pipette 10μL of both PA and FL onto separate plates of 4 agar types (TSA, LB, BG11, and LAKE) and spread.

This is the parent generation. 
8. PROPAGATE NEXT GENERATIONS:

8.1. From the lawns initially plated, by sterile loop pick up a single colony.

8.2. Select a plate of the same media.

8.3. Steak for isolation using a three phase streak pattern as shown below, sterilizing the loop in between each quadrant.

 

4. Label these plates "generation 1"
5. Continue until generation 3 is obtained.
6. Use generation 3 for testing.
TITLE: The relationship between chronic skin conditions and depression and how successful dermatologic treatment could improve patients’ mental health: An evidence-based systematic review protocol
AUTHORS: Aime Lee, Nami Watanabe, Emma Gregory, Cobi Desilva, Jenina Joyce Pali, Lindsey Leonhard, Samia Valeria Ozorio Dutra

[DESCRIPTION]
Psychodermatology is the interaction between the skin and the mind. Currently,
there is no standardized process for providing Dermatology patients with
depression screenings. We will conduct a systematic review to determine the value of providing patients who have chronic
skin conditions with a PHQ-9 screening prior to, during, and after treatment. 

*This protocol follows PRISMA-P guidelines and is provided as an attachment.* 

[STEPS]
TITLE: Rotarod test in rats
AUTHORS: eduard.bentea, María Sanchiz Calvo, Veerle Baekelandt

[DESCRIPTION]
Protocol for performing the rotarod test in rats. This test evaluated motor coordination and balance, and can be used in rat models with motor deficits, such as models of Parkison's disease. Rats with impaired motor function show a decreased latency to fall from the rotating rod.

[STEPS]
SECTION: Training
1. Bring cages to the behavioral room for at least 60 min prior to the test for habituation

SECTION: Training
2. Habituate rats on the rotarod for 5 min at 5 rpm, place back in case they fall

SECTION: Training
3. 5 min rest

SECTION: Training
4. Test rats for 1 min at 5 rpm

SECTION: Training
5. 5 min rest

SECTION: Training
6. Test rats for 1 min at 10 rpm

SECTION: Training
7. 5 min rest

SECTION: Training
8. Test rats for 1 min at 15 rpm

SECTION: Baseline
9. One day after training, perform baseline measurements
SECTION: Baseline
10. Bring cages to the behavioral room for at least 60 min prior to the test for habituation

SECTION: Baseline
11. Test rats at 4-40 rpm for a total duration of 5 min

SECTION: Baseline
12. 5 min rest

SECTION: Baseline
13. Test rats at 4-40 rpm for a total duration of 5 min

SECTION: Baseline
14. 5 min rest

SECTION: Baseline
15. Test rats at 4-40 rpm for a total duration of 5 min

SECTION: Test
16. At different timepoints during the experiment, evaluate changes in motor performance using the same protocol as during baseline
SECTION: Test
17. Bring cages to the behavioral room for at least 60 min prior to the test for habituation

SECTION: Test
18. Test rats at 4-40 rpm for a total duration of 5 min

SECTION: Test
19. 5 min rest

SECTION: Test
20. Test rats at 4-40 rpm for a total duration of 5 min

SECTION: Test
21. 5 min rest

SECTION: Test
22. Test rats at 4-40 rpm for a total duration of 5 min