eamag/protocols-with-steps huggingface dataset:
TITLE: CAMbank: cfDNA BCT Field Processing v1
AUTHORS: Eliah G Overbey, Krista A Ryon, jak, chm2042

[DESCRIPTION]
Field processing of cfDNA BCTs for the Cornell Aerospace Medicine Biobank (CAMbank).

Instructions for preserving: plasma and RBC Pellets.

[STEPS]
SECTION: Perform Venipuncture
1. After venipuncture, invert the tubes gently 8 to 10 times to fully mix tube anticoagulant with blood sample.

Store the tube upright at room temperature until centrifugation.
SECTION: Centrifuge Settings: Plasma Separation
2. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 20 minutes
speed: 300xg
SECTION: Centrifuge Settings: Plasma Separation
3. Place the cfDNA BCTs in the centrifuge. 
Place a protective cover over the swing buckets in case of tube leakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Separate Plasma
4. Carefully remove cfDNA BCTs from the centrifuge and inspect for separation of red blood vs plasma layers. 

Transfer cfDNA BCTs to a sanitized laminar flow hood. 
SECTION: Separate Plasma
5. Remove the upper plasma layer and transfer to a new 15mL conical tube. 
Cap the conical tubes and return to the centrifuge. 
SECTION: Aliquot Plasma
9. Carefully remove conical tubes from centrifuge. 
Transfer conical tubes to a sanitized laminar flow hood. 
SECTION: Aliquot Plasma
10. Using a P1000, carefully aliquot serum into 2D barcoded tubes at 500µL each. 

Return 2D tubes with plasma to the rack and place in the -80C freezer.
SECTION: Centrifuge Settings: Plasma Purification
6. Note: A swing bucket centrifuge is required. 

Set centrifuge:
acceleration: 9
deceleration: 9
temperature: RT
duration: 10 minutes
speed: 5000xg
SECTION: Centrifuge Settings: Plasma Purification
7. Place the conical tubes in the centrifuge. 
Place a protective cover over the swing buckets in case of tube leakage. 
Start the centrifuge. 
Stand by the centrifuge until the centrifuge reaches max speed. Listen for signs of imbalance or compromised tube integrity. 
SECTION: Freeze Red Blood Cell Pellets
8. Recap and freeze the cfDNA tubes to preserve DNA in the red blood cell pellet
TITLE: ATR FTIR Spectoscopy of Aqueous Cell Cultures
AUTHORS: Emrulla Spahiu, Senol Dogan, Jörg Schnauß, Mayda Gursel, Feride Severcan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">ATR-FTIR Spectroscopy of aqueous cell culture samples is explained step-by-step. Infrared spectra acquisition, processing and analysis is included briefly.</div></div>

[STEPS]
?. [Spectra acquisition]
Arrange the temperature of the working environment to
25 °C
?. [Spectra acquisition]
Clean the ATR crystal using a cellulose wipe and water-ethanol-water sequence. Scratching the crystal surface must be avoided.
?. [Spectra acquisition]
Leave the crystal to air-dry
?. [Spectra acquisition]
Select “Scan and Instrument Setup” and input sample name, scan range (650-4000 cm-1), scan number (128), and  spectral resolution (2 cm-1). This step needs optimization for specific sample being used.
?. [Spectra acquisition]
Scan the backgroung spectrum to overcome the probable atmospheric interference that can be triggered by H2O and CO2 molecules present in the air, subtracted automatically by the software.
?. [Spectra acquisition]
Additionally, record PBS as sample (subtract this manually from each spectrum)This eliminates interference of PBS and flattens the water band located around 2125 cm -1
?. [Spectra acquisition]
Put   cell samples and let to dry with a mild N2 flux for  . This allows cells to settle on the crystal.
1 µl
?. [Spectra acquisition]
Scan the sample. Process takes about  to finish.As the scanning process goes on, one must be sure that the quality check sign is everytime green.
?. [Spectra acquisition]
Scan each sample under the same conditions in three independent scans (the average constitutes a replicate n). Replicates (n=5) are used in data analysis. This minimizes intra-sample variability and eliminates possible variation that might arise from experimental conditions.
?. [Spectral pre-processing]
The avarage of triplicates is taken manually.
?. [Spectral pre-processing]
13-point Savitzky-Golay smoothing
?. [Spectral pre-processing]
Baseline correction at 3800-2750-1800-900 cm-1points.
?. [Principal Component Analysis (PCA)]
Import spectra as data file into
?. [Principal Component Analysis (PCA)]
Define the data sets to 3000-650 cm-1region.
?. [Principal Component Analysis (PCA)]
Make the PCA Model.Define the regions with high variation using the Loadings plots.Check how the replicates are grouping and if there are outliers in Scores plots.
?. [Spectral Calculations]
Peak labelling can be done automaticaly. For increased precision we measured manually:Measure the center of weight of each band. Read the frequency.Compare the peaks you obtained, to the literature:Make an excel file of your peaks and their assignment to the functional groups. For side-peak band assignment, the second derivative spectrum is used.
?. [Spectral Calculations]
Concentration studies are carried out by integrating the curves under the peaks.
?. [Spectral Calculations]
For conformational alteration studies, bandwidth measurements are done manually using the 75% height of the peaks.
TITLE: Fungal DNA Extraction from Fungal Plate
AUTHORS: You Li, Demian F Gomez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to do fungal DNA extraction from fungal plate. </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]
TITLE: How to check the integrity of a set of files uploaded to AWS S3
AUTHORS: Sonia García-Ruiz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol contains the instructions to verify the integrity of any small or large file uploaded to AWS S3. </div><div class = "text-block">This protocol has been successfully tested on:</div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div></div>

[STEPS]
?. Clone the s3md5 repo:$ git clone https://github.com/antespi/s3md5.git
?. Grant execution access to the s3md5 script file.$ chmod 755 ./s3md5/s3md5
?. Clone the aws-s3-integrity-check repo:$ git clone https://github.com/SoniaRuiz/aws-s3-integrity-check.git
?. Move the 's3md5' folder within the 'aws-s3-integrity-check' folder:$ mv ./s3md5 ./aws-s3-integrity-check
?. The 'aws-s3-integrity-check' folder should look now similar to the following structure:$ ls ./aws-s3-integrity-checktotal 16 -rw-r--r-- 1 your_user your_group 3573 date README.md -rwxr-xr-x 1 your_user your_group 3301 date aws_check_integrity.sh drwxr-xr-x 2 your_user your_group 4096 date s3md5
?. Execute the 'aws-check-integrity.sh' bash script following the instructions below:$ aws_check_integrity.sh   Usage :: local path to the folder that contains all files previously uploaded to AWS. For example: /local-path/raw-data-folder/. : name of the S3 bucket containing the files uploaded to AWS that we want to check their integrity. For example: 'bucket-name'.: folder name on the S3 bucket that contains all files to be checked. The name of this folder should be the same indicated on the  parameter. For example: raw-data-folder/. In case there is not any root folder, this parameter will be substituted by a slash (/), which will indicate the root path.Example:$ aws_check_integrity.sh /local-path/raw-data-folder/ bucket-name raw-data-folder/
?. The execution of this script will:Loop through all files stored on /local-path/raw-data-folder/.Per each file, the script will check its size. In case the object found is a directory, it will just continue looping through its child directory files.If the file size is smaller than 8MG, the script will generate a simple MD5 digest value.If the file size is bigger than 8MG, it will make a request to the s3md5 script, which will apply the same algorithm as AWS does: it will split the file into 8MG small parts, generate the MD5 hash corresponding to each small part and generate the final MD5 digest number from the total set of individual MD5 hashes.Retrieve the ETag value from the corresponding file stored on the S3 bucket.Compare the retrieved ETag value with the integrity number generated by the script.If both numbers are identical, the script will confirm the integrity of the file stored on the S3 bucket. Otherwise, the script will generate an error. In any case, the result will be stored within a log file whose name will follow the following pattern: S3_integrity_log.timestamp_bucketname.txt. The log file will be stored within a folder called 'logs'. In case this folder doesn't exist, it will be automatically created by the script in the same path in which it has been executed.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Goal: Identify 16S rRNA gene metagenomic fragments and create assembled full-length 16S rRNA sequences from fragments.<br />Note 1 - As is this case with most bioinformatic processes, this is one of many possible methods, but has had successful results in the past<br />Note 2 - This method is best applied to sequences that have been subjected to quality control as in https://www.protocols.io/view/Basic-Illumina-Sequence-Quality-Control-d4e8td

[BEFORE_START]
**There are many tools available for this process - this is one example that has been used effectively**<br />Required software:<br />IDBA-UD v. 1.1.1 - http://i.cs.hku.hk/~alse/hkubrg/projects/idba_ud/<br />Meta-RNA - http://weizhong-lab.ucsd.edu/meta_rna/<br />HMMER v3.1b2 - http://hmmer.janelia.org/<br />MetaRNA_to_FastQ.py - https://github.com/bjtully/BioData/tree/master/Various_Tools<br />Biopython - http://biopython.org/DIST/docs/tutorial/Tutorial.html#htoc4<br />EMIRGE - https://github.com/csmiller/EMIRGE<br />Cython<br />pysam<br />scipy/numpy<br />usearch (<a href="http://www.drive5.com/usearch/" target="_blank">www.drive5.com/usearch/</a> -- tested with usearch version 6.0.203)<br />samtools (<a href="http://samtools.sourceforge.net/" target="_blank">http://samtools.sourceforge.net/</a> -- tested with verison 0.1.18)<br />bowtie (<a href="http://bowtie-bio.sourceforge.net/index.shtml" target="_blank">http://bowtie-bio.sourceforge.net/index.shtml</a> -- tested with version 0.12.7 and 0.12.8)

[STEPS]
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TITLE: Autophagosome live cell imaging
AUTHORS: Dan Tudorica, bishal.penn

[DESCRIPTION]
For quantification of PINK-PARKIN mitophagy

[STEPS]
SECTION: Cell Culture
1. Seed Parkin-expressing HeLa cells for ~80% confluence on day of transfection on tissue-culture coated glass slides, suitable for use on a confocal microscope.
SECTION: Cell Culture
2. On day of transfection, transfect pCIG2-LAMP1-mNeon Green, pHaloTag-LC3B, and an autophagy regulating protein tagged with mcherry via lipofectamine 3,000 (ThermoFisher). Briefly, dilute plasmids in Opti-mem reduced serum medium and add P3000 reagent in accordance with manufacturer instructions. Also dilute lipofectamine 3000 reagent in optimem, then mix the dilute plasmid and lipofectamine solutions together. Incubate for ~10 minutes, then add dropwise to adhered cells.
SECTION: Cell Culture
3. Allow cells to recover overnight
SECTION: Cell Culture
4. On day of experiment swap cells into growth medium containing 10 µM Oligomycin A and 5 µM Antimycin A. Return to incubator for 4 H
SECTION: Imaging and Analysis
5. When ready to image, swap medium with Leibovitz’s L-15 medium (Gibco 11415064) supplemented
with 10% fetal bovine serum and 1% Glutamax along with Antimycin A and
Oligomycin A. 
SECTION: Imaging and Analysis
6. Calibrate laser power and exposure for best image quality. Keep these parameters consistent across treatments or conditions. Collect Z stacks of each target cells.
SECTION: Imaging and Analysis
7. To analyze, threshold LC3 and LAMP1 channels, then binarize using Yen thresholding in
ImageJ. To quantify area of colocalization, use the ‘AND’ function from the Image
Calculator function. Then calculate area of colocalization using Analyze
Particles.
TITLE: Changes in spinal stiffness with chronic thoracic pain: correlation with pain and muscle activity
AUTHORS: Isabelle Pagé, François Nougarou, Arnaud Lardon, Martin Descarreaux

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The main goal of this project is to assess the reliability of thoracic spinal stiffness. To achieve this, 25 heartily participants (no significant thoracic pain in the past year) and 25 participants with chronic thoracic pain will participate in 2 experimental sessions separated by 24 to 48 hours. These sessions will occur at the Neuromecanic and Motor Control Laboratory of the Université du Québec à Trois-Rivières (Trois-Rivières, Québec, Canada). During these sessions, spinal stiffness will be measured at four thoracic spinal levels (T5 to T8) in a randomized sequence via a mechanical device using a servo-linear motor. The device indenter (metallic cylindrical cover with a rubber of 2 cm diameter) will gradually apply 45 N to the spinous process. The muscle activity and displacement data will be recorded during the procedures. </div></div>

[STEPS]
TITLE: Manual H&E Staining
AUTHORS: Jia Ren Lin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The hematoxylin and eosin (H&E) stain is the standard used for microscopic examination of fixed tissue specimens. This procedure establishes a consistent process for the manual preparation of H&E-stained slides from FFPE tissue specimens. H&E staining is compatible with, and frequently performed after tissue-based cyclic immunofluorescence (t-CyCIF), enabling conventional histopathology review to be combined with fluorescence imaging.</div></div>

[STEPS]
?. [Post-CyCIF Decoverslipping]
After the final imaging step of CyCIF (or IF), soak slides in 1X PBS for  and gently detach coverslips from slides using slide forceps.
0 Room temperature
?. [Manual H&E Staining]
Remove slides and place on the stain box. Add between  and  Hematoxylin solution and let incubate for .
200 µl
400 µl
0 Room temperature
?. [Manual H&E Staining]
Wash slides by placing them into the staining jar with running tap water for .
0 Room temperature
?. [Manual H&E Staining]
Put washed slides back to the staining box. Add between  and  Eosin Y solution and incubate for .
200 µl
400 µl
0 Room temperature
?. [Manual H&E Staining]
Wash slides by placing into the staining jar with running tap water for at least  until all the excess red Eosin color is gone.
0 Room temperature
?. [Manual H&E Staining]
Air dry slides completely (at least ), mount slides with  Permount solution, and gently place coverslips. Slides can be imaged after .
80 µl
?. [Post-CyCIF Decoverslipping]
Wash slides 1-2 times in the staining jar with tap water .
0 Room temperature
TITLE: Psychometric evaluation of the Korean version of the Hepatitis B Quality of Life Questionnaire
AUTHORS: Yeonsoo Jang, Sang Hoon Ahn, Kyunghwa Lee, Jiyeon Lee, Jeong Hyun Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Purpose: The purpose of this study was to conduct a psychometric evaluation of the Korean version of the Hepatitis B Quality of Life Questionnaire (K-HBQOL), which is designed to assess the quality of life of patients with the hepatitis B virus (HBV).</div><div class = "text-block">Methods: The K-HBQOL was developed by converting the original English version to Korean using a back-and-forth translation method. The translated questionnaire was distributed to 168 adults with HBV. Descriptive statistics were used to summarize the demographic characteristics of these participants. Confirmatory factor analysis and exploratory principal components analysis (CFA and PCA) were performed, applying varimax rotation and using Kaiser’s eigenvalue-greater-than-one rule to examine the factor structure. The minimum average partial rule was also used to identify the number of components.</div><div class = "text-block">Results: The original factor model was not confirmed by CFA in this sample. Six components (Psychological Well-being, Stigmatization, Anticipation Anxiety, Weakness, Vitality, and Vulnerability) were extracted by PCA, with 69.11% of the total variance explained. In this process, one component present in the original factor model (Transmission) was not found, while another component (Weakness) was extracted.</div><div class = "text-block">Conclusions: This study revealed the psychometric characteristics of the Korean version of the HBQOL for patients with hepatitis B. We suggest a study with a larger sample is needed to evaluate validity and reliability of the K-HBQOL for other Korean populations.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p>This protocol uses an adapted version of Omega Bio-Tek’s E.Z.N.A. Bacterial DNA Kit. In new versions of the kit BTL Buffer has been renamed TL Buffer, BDL Buffer has been renamed BL Buffer, and HB Buffer has been renamed HBC Buffer.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Melanophores (also called melanocytes) are chromatophores (pigment-containing epithelial cells) that contain black or dark brown pigment granules. The aggregation or dispersal of these cells is under hormonal or nervous system control; these responses permit the animal to display rapid variations in color in response to environmental stimuli, including background color and stressful stimuli. This protocol was developed at Laboratório de Neurociências e Comportamento "Frederico Guilherme Graeff", at the Universidade Federal do Sul e Sudeste do Pará (UNIFESSPA), Marabá/PA, Brazil, to assess melanophore aggregation or disperal responses in live, anesthetized adult zebrafish. The protocol is intended to be easily applied, reproducible, and of low cost.</p>

[STEPS]
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TITLE: DNeasy PowerSoil Pro Kit modification for soil fungal community barcoding
AUTHORS: Tyler W. d'Entremont, Stephanie N. Kivlin

[DESCRIPTION]
Here we provide a modified protocol for use with the Qiagen PowerSoil Pro Kit that has been used with great success for obtaining fungal DNA from field collected soil samples for downstream barcoding.

[STEPS]
1. Centrifuge the PowerBead Pro Tubes briefly to ensure contents have settled to the bottom.
2. Weigh out 250 mg of soil and add to the Powerbead Pro Tube. Add 750 µL of Solution CD1 and 50 µL of Solution ATL (ordered seperately from the kit contents). Vortex to homogenize.
3. Place samples in a Biospec Mini-beadbeater (or equivalent) at 3800 rpm for 1 min.
4. Remove samples and place on ice for 5 min.
5. Repeat Step 3.
6. Centrifuge tubes at 15,000 x g for 1 min.
7. Transfer supernatant (~500-600 µL) to a 2 mL microcentrifuge tube, avoiding debris as much as possible. Keep samples on ice or in a cold microcentrifuge tube block.
8. Add 200 µL of Solution CD2 and vortex for 5 sec.
9. Centrifuge tubes at 15,000 x g for 1 min.
10. Transfer up to 700 µL to a new 2 mL microcentrifuge tube, avoiding the pellet at the bottom.
11. Add 600 µL of Solution CD3 and vortex for 5 sec.
12. Pipette 650 µL of lysate onto the MB Spin Column membrane and centrifuge at 15,000 x g for 1 min.
13. Discard the flow-through and repeat Step 12 with the remaining sample.
14. Place the MB Spin Column in a new 2 mL collection tube and add 500 µL of Solution EA to the MB Spin Column. Centrifuge at 15,000 x g for 1 min.
15. Discard flow-through and using the same collection tube, add 500 µL of Solution C5 to the MB Spin Column. Centrifuge at 15,000 x g for 1 min.
16. Place the MB Spin Column in a new 2 mL collection tube and centrifuge at 16,000 x g for 2 min.
17. Place the MB Spin Column in a 1.5 mL Elution Tube and pipette 60 µL of Solution C6 (or water), ensuring it is placed directly onto the filter membrane. Allow tubes to sit for 5 min at room temp.
18. Centrifuge tubes at 15,000 x g for 1 min, then discard MB Spin Column. DNA is now ready and should be stored at -20oC.
TITLE: Upper limb superficial venous percussion as a sign of anatomical location and venous permeability
AUTHORS: Pedro Coelho N Diogenes, Aline Naiara Azevedo da Silva, Fausto Pierdoná Guzen, Marco Aurelio de Moura Freire, José Rodolfo Lopes de Paiva Cavalcanti

[STEPS]
?. The upper limb being examined was garrotted.
?. The anatomical sites of the main four superficial venous segments of the upper limbs was examined separately (cephalic vein in forearm, cephalic vein in arm, basilic vein in forearm and basilic vein in the arm).
?. Each segment was struck distally with forefinger of the dominant hand with the garrotted limb. Small impacts were generated on the patient's skin in the anatomical path of the superficial venous segment with the index finger of the dominant hand of the examiner at a point distal to the patient's limb, while the palmar face of the of the examiner's non-dominant hand was located at a proximal point on the way of this main superficial venous segment (about 15 cm away).
?. In the non-dominant hand of the examiner (proximal), the proximal perception of the transmitted distal wave impacts caused by percussion (impacts) with the index finger of the dominant (distal) hand resulted in a positive maneuver on the venous segment examined.
?. The maneuver was declared negative after at least 10 impacts generated by the dominant hand (distal) and not transmitted to the proximal hand of the examiner (about 15 seconds of attempts).
?. A video demonstration is availeable at this following dropbox link https://www.dropbox.com/s/lu7m5jb2y0naxtm/percussao.mp4?dl=0