eamag/protocols-with-steps huggingface dataset:
TITLE: Rehabilitation interventions for impaired handwriting in people with Parkinson’s disease: a protocol for a scoping review
AUTHORS: Andrea Gardoni, Elisabetta Sarasso, Federica Agosta, Massimo Filippi, Davide  Corbetta

[DESCRIPTION]
Handwriting abnormalities in people with Parkinson’s disease include several dynamic and kinematic features such as increased writing stroke duration, reduced velocity and altered fluency of the writing strokes. “Dysgraphia” is a term that includes all the possible handwriting alterations and the “micrographia”, that defines an impairment of a fine motor skill manifesting mainly as a progressive or stable reduction in amplitude during a writing task, is only one of the characteristics of parkinsonian handwriting. Few studies investigated the effects of a rehabilitative training on handwriting or used measures of handwriting as an outcome in people with PD.
We aim to conduct a scoping review to answer the following question: “What are the effects of a rehabilitative program on handwriting in people with PD?” 

[STEPS]
SECTION: Search strategy
1. The search strategy was runned in April 2022
SECTION: Study records management, selection and data collection
2. 
SECTION: Risk of bias assessment
3. Requested time will depend on how many studies will be included
SECTION: Data synthesis
4. Requested time will depend on how many studies will be included
TITLE: Flow Cytometry ECS Surface Antigens
AUTHORS: Michael Betts, Gregory Golden

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">High-parameter flow cytometry enables identification and characterization of a wide range of cell populations within a biological sample. Analysis of cell-surface epitopes using a collection of fluorophore-labeled antibodies, or extracellular stain (ECS), sufficiently labels population-identifying markers for further analysis with flow cytometry. Importantly, ECS can be applied to cryo-preserved cell suspensions recovered in tissue culture media, enabling samples to be conveniently analyzed after collection and proper cryopreservation. However, consistent cryopreserved sample recovery and ECS procedures are critical to data comparison across multiple experiments. Herein, we describe a standardized protocol for cryopreserved sample recovery and ECS procedures for cell-surface epitope labeling. </div></div>

[STEPS]
?. [Procedure]
Thawing and Restinga. Pre-warm R10 media in a  water bath. b. Thaw samples in-vial using a  water bath.c. Add thawed cells to  of R10, then spin cells at 500 xg for 5 min.d. Resuspend cell pellet in  of R10 and count cells. e. Rest cells at least 3 hours (up to overnight) at 2x106 cells/mL in R10 medium + 1 µL/mL DNAse I at  , 5% CO².Note: during resting, prepare  antibody cocktail master mix. Adjust volume of ECS for  per test with FACS buffer.f. After resting, add PBS up to  or  (whichever is closer, rounding up) to cells and transfer to  or  conical tube.g. Spin cells at 500 xg for 5 minutes at room temperature (RT).h. Resuspend cells in PBS to 1x107 cells/mL and count. If cells are too dilute, re-spin cells and resuspend at 1x107 cells/mL. Transfer  of cells (2x106cells) into each well of a V-bottom 96 well plate.
37 °C
37 °C
14 mL
3 mL
37 °C
50 µl
15 mL
50 mL
15 mL
50 mL
200 mL
?. [Procedure]
Viability and extracellular staining (ECS)a. Spin plate at 500 xg for 5  minutes at RTb. Using a multichannel pipette, carefully remove the supernatant.c. Using a multichannel pipette, add  of PBS to well.d. Add  of 1:60 Aqua viability dye directly to cell pellet and resuspend cells.e. Incubate for 10 minutes at RT in the dark.f. Add  of ECS antibody cocktail to cells and incubate for 20 minutes at RT in the dark (prepare 1% PFA fixation buffer in the meantime).g. Add  of FACS buffer to each well and spin plate at 500 xg for 5 minutes. h. Discard supernatant and fix cells with  of 1% PFA.i. Run samples on flow cytometer.
45 µl
5 µl
50 µl
100 µl
200 µl
TITLE: Inhibition immunoassay of the HIVgp120_anti-HIVgp120 (Ab-1) reaction by  anti-anti-idiotypic-HIVgp120 antibody (Ab-3). 
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. Ninety six well polystyrene microplate (U-shaped bottom, Sigma-Aldrich Co, St Louis Missouri) is coated with 50 µl/well of 1 ng/µl solution of gp-120 peptide, fragment 254-274 (Sigma) in carbonate-bicarbonate buffer pH 9.6 (Sigma) for 4 hours at 37°C.
?. The microplate is then washed 4 times with PBS Tween-20.
?. The microplate is blocked with 3% non-fat milk in PBS, 25 µl/well, 1h, RT.
?. Triplicates of serial doubling dilutions of cat serum diluted in PBS-3% non-fat milk, pH 7.4 are added to the microplate which is then incubated for 90 min at 37°C.
?. The microplate is washed 4 times with 150 µl/well of PBS-Tween 20, and 25 µl of a water soluble fraction (WSF) containing anti-HIV-gp120 antibodies (titer =1:1024) is added to each well.
?. After adding WSF and it follows an incubation period (90 min, 37°C).
?. The microplate is washed 4 times and 25 µl of a 1:30,000 dilution of rabbit anti-chicken IgY-HRP (Sigma-Aldrich Co) is added to each well.
?. The microplate is then incubated at 37°C for 1 h.
?. The microplate is washed with PBS-Tween 20, 4 times.
?. Add 25 µl of TMB to the each well of the microplate and incubate the plate in the dark for 15 min.
?. The reaction was stopped with 3M H2SO4.
?. The microplate is read at 450 nm in a microplate reader.
TITLE: Crude extraction of Xylella fastidiosa from infected leaves
AUTHORS: Niels Appelman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol can be used to (crudely) extract Xylella fastidiosa from infected leaves.</div></div>

[STEPS]
?. Pick leaves from plants infected with X. fastidiosa
?. With a scalpel, remove the first 2 cm of the central nerve of the plant was removed.Note: X. fastidiosa is located in the xylem in this nerve. Including mesophyll will reduce the yield.
?. Crush the central nerve and extract bacteria with 50 ml of 0.01 M potassium phosphate buffer.
?. Drain the extract in a 50 ml conical centrifuge tube.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The importance of fungal species differentiation is critical for plant pathology research and disease control purposes. Traditionally, fungal species have been identified by a range of cultural and morphological characteristics, such as conidial morphology, presence or absence of setae, fungicide sensitivity, and colony color and growth rate. Although valuable, these criteria alone are not always adequate as morphological characteristics may vary under different environmental conditions. Among other techniques, sequence analyses of the internal transcribed spacer (ITS) regions, including the 5.8S rDNA, has been used extensively to identify fungal species. This region is the most widely sequenced DNA region of fungi and has been employed as the universal fungal barcode sequence. It is very useful for molecular systematics at species to genus level and within species.</p>
<p>This protocol is useful for a primary molecular and accurate identification of any fungal species using the sequence analyses of the ITS-5.8S rDNA region</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: RT–PCR protocol for the detection ORF1 11288–11296 deletion (NSP6 106-108del) in SARS-CoV-2 genome
AUTHORS: Nikita Yolshin  , Artem  Fadeev, Andrey  Komissarov

[DESCRIPTION]
ORF1 deletion is observed in some new SARS-CoV-2 lineages and is lineage defining for B.1.1.7 and P.1. Biological effect of this mutation is still unknown. Developed RT-PCR was tested on numerous samples of lineages B.1.1.7, B.1.351, P.1 (validated by sequencing) and samples without this mutation with no false-postive results.

  
 


[STEPS]
1. 
Order oligonucleotides with following sequences:

 
     Name      Sequence   (5’ – 3’)        ORF1-del-F      GGTTGGATATGGTTGATACTAGTTTGAAG        ORF1-del-R      TGTCAAGACATTCATAAGTGTCCACA        ORF1-del-P      Cy5.5-ACTGTGTTATGTATGCATCAGCTGTAGTGTTACTAATCC-BHQ3   
 
     2X RT-PCR Buffer      12.5 μl            25X RT-PCR Enzyme Mix      1 μl            ORF1-del primers      600 nM each           ORF1-del probe      400 nM           RNA template      5 μl            Nuclease-free water      to 25 μl      
 
2. Briefly vortex and centrifuge reagents before use.
Prepare the PCR reaction mixture following the specifications below:
 
     2X RT-PCR Buffer      12.5 μl            25X RT-PCR Enzyme Mix      1 μl            ORF1-del primers      600 nM each           ORF1-del probe      400 nM           RNA template      5 μl            Nuclease-free water      to 25 μl      
 
Use SARS-CoV-2 positive samples as the template. In the case of screening of unknown samples use multiplex RT-PCR with some oligonucleotides for detection SARS-CoV-2 as a control reaction. This primer set for SARS-CoV-2 detection can be added to the test without decrease of sensitivity of the ORF1 deletion test:

 
     Name      Sequence (5’ – 3’)            2-SARS-CoV-2-ORF-1-F      AGAGCTATGAATTGCAGAC                      2-SARS-CoV-2-ORF-1-R      GGGAAATACAAAATTTGGACA            2-SARS-CoV-2-ORF-1-P      FAM-AATTGGCAAAGAAATTTGACACCTTCA-BHQ-1      
 
 

 


 
     Name      Sequence (5’ – 3’)            2-SARS-CoV-2-ORF-1-F      AGAGCTATGAATTGCAGAC                      2-SARS-CoV-2-ORF-1-R      GGGAAATACAAAATTTGGACA            2-SARS-CoV-2-ORF-1-P      FAM-AATTGGCAAAGAAATTTGACACCTTCA-BHQ-1      
 

 
3. Perform the amplification in a general thermocycler with appropriate temperature profile:

  

4. Interprete the results: detection of fluorescence probe “ORF1-del-P” (Cy5 channel in our case) means presence of ORF deletion 11288–11296
TITLE: Study Protocol Document for PONE-D-21-18427R2 (Prevalence and Predictors of GMD among PLWH on cART)
AUTHORS: Wondmagegn  T. Tadesse,   Wondwossen Amonge, Eleni Akllilu,   Ephrem Engidawork

[DESCRIPTION]
This is a document that states about sample size calculation, study participant recruitment, interview and medical record data extraction sheet, sample collection, processing and storage until lab analysis. Moreover, it states about how Glucose Metabolism Disorders (GMD) is determined with specific forms of GMDs.   

[STEPS]
TITLE: FACS for CD45+ immune cells
AUTHORS: Connor Monahan

[DESCRIPTION]
This protocol details FACS of intestinal immune cells (CD45+).

[STEPS]
SECTION: Procedure
1. Resuspended cells isolated from the small intestine with 400 µL Fc block (BD Biosciences,
Cat# 553142; Franklin Lakes, NJ), 1:200 in FACS buffer.
SECTION: Procedure
2. Incubate for 20 min on ice.
SECTION: Procedure
3. Wash cells in 500 µL FACS buffer, pellet at 1500 rpm, 2 min, then resuspend in 600 µL of FACS buffer with 1:100 CD45 BUV395 antibody (BD Biosciences, Cat# 564279; Franklin Lakes, NJ).
SECTION: Procedure
4. Incubate on ice for 30 min.

SECTION: Procedure
5. After 30 minutes, wash cells in 500 µL FACS buffer.

SECTION: Procedure
6. Resuspend in 500 µL of FACS buffer containing propidium iodide (BioLegend, Cat# 421301; San Diego, CA) at 1:10,000.

SECTION: Procedure
7. Sort viable, CD45+ cells on a Sony MA900 Multi-Application Cell Sorter.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Purpose: Dry Eye Disease (DED) is part of several conditions, including Sjögren’s syndrome (SS) and no single test to diagnosis DED. The present study intends to evaluate whether a set of signs and symptoms of DED can distinguish: a) SS from other non-overlapping systemic diseases related to DED; b) primary and secondary SS.
Methods: 182 consecutive patients with DED were evaluated under five groups: SS, graft-versus-host disease (GVHD), Graves' orbitopathy (GO), diabetes mellitus (DM), glaucomaunder treatment with benzalkonium chloride medications (BAK). Twenty-four healthy subjects were included as control group (CG). The evaluation consisted of Ocular Surface Disease Index (OSDI), Schirmer test (ST), corneal fluorescein staining (CFS) and tear film break up time (TFBUT). Indeed, a subset of DED patients (n=130), classified as SS1, SS2 and nonSS (NSS) by the American-European Criteria were compared. Quadratic discriminant analysis (QDA) classified the individuals based on variables collected. The area under Receiver Operating Characteristics (ROC) curve evaluated the classification performance in both comparisons.
Results: Comparing SS with other diseases, QDA showed that the most important variable for classification was OSDI, followed by TFBUT and CFS. Combined, these variables were able to correctly classify 62.6% of subjects in their actual group. At the discretionof the area under the ROC curve, the group with better classification was the control (97.2%), followed by DM (95.5%) and SS (92.5%). DED tests were different among the NSS, SS1 and SS2 groups. The analysis revealed that the combined tests correctly classified 54.6 % of the patients in their groups. The area under the ROC curve better classified NSS (79.5%), followed by SS2 (74.4%) and SS1 (69.4%). 
Conclusions: Diseases that causes DED, and also SS1, SS2 and NSS are distinguishable conditions, however a single ocular tools was not able to detect the differences among the respective groups.

[STEPS]
TITLE: Purification of GST-TEX264
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification and analysis of GST-TEX264. 

[STEPS]
SECTION: Purification procedure
1. To purify GST-TEX264, fuse the cytosol-exposed domain of TEX264 (28-313aa) to a N-terminal synthesize gene GST-tag by Genscript and clone into a pGEX-4T1 vector. Plasmid also available from Addgene. 

SECTION: Purification procedure
6. Collect the supernatant and incubate with pre-equilibrated Glutathione Sepharose 4B beads (GE Healthcare) for 120 min at 4 °C with gentle shaking to bind GST-TEX264. 

SECTION: Purification procedure
5. Sonicate the cell lysates twice for 30 s and clear by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

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

Lysis Buffer:
 
 
 
SECTION: Purification procedure
3. Once the cells reach 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 procedure
2. After the transformation of the pGEX-4T1 vector encoding GST-TEX264 in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow 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 procedure
12. Analyze fractions by SDS-PAGE and Coomassie staining. Pool the fractions containing purified GST-TEX264.
SECTION: Purification procedure
11. Elute the proteins with SEC buffer. 

SEC Buffer:
 
 
 
SECTION: Purification procedure
10. Pool the supernatant fractions, filter through a 0.45 µm syringe filter, concentrate with 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 

SECTION: Purification procedure
9. To collect the supernatant, collect the beads by centrifugation. Wash the beads twice with 4 mL of wash buffer, and collect the supernatant. 

SECTION: Purification procedure
8. Incubate the beads 120 min with 4 mL of 50 millimolar (mM) reduced glutathione dissolved in wash buffer at 4 °C, to elute GST-TEX264 from the beads. 

Wash Buffer:
 
 
 
SECTION: Purification procedure
7. Centrifuge the samples to pellet the beads and remove the unbound lysate. Then wash the beads twice with wash buffer, once with high salt wash buffer, and two more times with wash buffer. 

Wash Buffer:
 
 
 
Salt wash Buffer:
 
 
 
SECTION: Purification procedure
13. After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen. 

SECTION: Purification procedure
14. Store the proteins at -80 °C.
TITLE: Tissue collection and extractions for RNA-seq
AUTHORS: David Lowry

[STEPS]
?. Pour liquid nitrogen into a polystyrene cooler or another container that can safely hold liquid nitrogen.
?. Remove tissue from plant and place in a tube. Make sure to clean scissors or foreceps with ethanol prior to collections.
?. Immediately plunge tube into liquid nitrogen (transcription profiles change rapidly, so do this as fast as possible).
?. Transfer tubes into a cooler containing dry ice for transport to a -80C freezer. Store at -80C until ready to extract.
?. Extract tissue with a Spectrum Plant Total RNA Kit (Sigma-Aldrich). The Lowry lab has achieved consistently high yields of RNA from Mimulus guttatus using this kit. https://www.sigmaaldrich.com/catalog/product/sigma/strn50?lang=en&region=US&gclid=CjwKCAiA__HvBRACEiwAbViuU4EOr5CpJNBCqRf-923Uw8ZrDfSQ6bRXNHIhX4t6lKE7jA5AAHO5UxoCtP0QAvD_BwE
TITLE: Catalase test for bacterial identification
AUTHORS: lydiariver  

[DESCRIPTION]
The catalase enzyme is found in most aerobic and facultative anaerobic bacteria that contain cytochrome, being the main exception Streptococcus. Organisms that do not have the cytochrome system also lack the enzyme catalase and therefore cannot break down hydrogen peroxide.


[STEPS]
1. Transfer 2-4 colonies of the bacterial growth in blood agar plates to a glass
slide using a sterile plastic loop, make circles, and let it dry.
 

2. Place a drop of hydrogen peroxide (H 2 0 2 ) on the glass slide with an
eyedropper.
 

3. Observe immediate results.
Positive: The oxygen released will be observed as a formation of bubbles.
Negative: No or very few bubbles produced.
 

4.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Experiment purpose is to monitor the time-course of a large-scale infection of host cyanobacteria by phage under variable media conditions and obtain samples for proteomic and transcriptomic analysis.</strong></p>
<p> </p>
<p><strong>9 Hourly Timepoints: 0, 1, 2, 3, 4, 6, 8, 10, 14</strong></p>
<p> </p>
<p><em>Sampling is to collect supernatant fractions to determine total dissolved phosphate and soluble reactive phosphate (TDP/SRP) for analysis of inorganic phosphate concentration and phosphate in dissolved organic material (DOM); and to collect pellets to isolate RNA for transcriptomics and proteins for proteomics.</em></p>

[STEPS]
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TITLE: PCR-based assay for genotyping of the slick mutation in cattle
AUTHORS: Elizabeth Jannaman, M. Sofia Ortega, Tad S. Sonstegard, Peter J. Hansen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Several mutations exist in the prolactin receptor gene (PRLR) of cattle that cause a short, sleek hair</div><div class = "text-block">coat.. The mutation is dominant so slick-haired cattle can have either one or</div><div class = "text-block">two copies of the mutation. The assay described here detects the most-well studied mutation (SLICK1)</div><div class = "text-block">that causes translation of a truncated PRLR of 461 amino acids. Originally described in Senepol cattle,</div><div class = "text-block">this mutation has also been introduced into Holsteins in Puerto Rico, Florida and elsewhere. Note that</div><div class = "text-block">the assay will not detect other mutations in PRLR that also cause the slick phenotype.</div></div>

[STEPS]
?.
TITLE: Artificial Cerebrospinal Fluid V (ACSF.V)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to prepare Artificial Cerebrospinal Fluid (V). Artificial Cerebrospinal Fluid (V) is used during surgical procedures to maintain tissue hydration and minimize tissue damage, adhesion formation and infection.</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]
Protocol for Dephosphorylation of 5&acute;-ends of DNA using CIP in Restriction Enzyme Reaction. Uses the Calf Intestinal&nbsp;Alkaline Phosphatase &nbsp;(CIP - M0290)

[STEPS]
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TITLE: SOP for DSS kinetics studies
AUTHORS: Malu G Tansey

[DESCRIPTION]
SOP for DSS kinetics studies

[STEPS]
SECTION: PROTOCOL:
2. Weigh each mouse from a cage individually in small boxes. Record their weights in grams to the nearest tenth for that day.
SECTION: PROTOCOL:
3. Keep each mouse in their own box until stools are passed. For a healthy mouse, this should occur quickly. A mouse can be scruffed to expedite this if needed.
SECTION: PROTOCOL:
4. Once stool is passed, return the mouse to its home cage. While doing this, inspect the mouse’s rectum and look for bleeding and/or smeared stool. These features reflect more severe colitis and will be used in DAI scoring later.
SECTION: PROTOCOL:
5. With an applicator stick, pick up the stool and smear it onto one of the designated areas of a slide. Observe the appearance and overall feel of the stool as it spreads onto the slide. Also, observe whether blood is visible in the smear.
SECTION: PROTOCOL:
6. Open the other side of the slide and observe whether there is any soaking through from the side to which the stool was applied.

SECTION: PROTOCOL:
7. Score the stool consistency according to the rubric below:
 

SECTION: PROTOCOL:
8. Add two drops of developer solution to the backside of the card and to the quality control panel below.
SECTION: PROTOCOL:
9. After at least 30s, verify that the positive control dot is blue and the negative control dot is not.
SECTION: PROTOCOL:
10. Look back to the side to which the stool was applied. If you can see blue, consider the sample as positive.
SECTION: PROTOCOL:
11. Then, score the fecal blood according to the rubric below:
 

SECTION: PROTOCOL:
12. Score the weight loss according to the rubric below:
 

SECTION: PROTOCOL:
13. For statistical analysis and data visualization, sum all three scores into one total DAI score.
SECTION: BEFORE STARTING:
1. Ensure you have garnered the appropriate approval from your institution’s regulatory body over animal use in research to conduct experiments with DSS, and comply with any additional instructions they may provide to ensure your research animals are being properly cared for. 

In cages with multiple animals, ensure animals have unique identifiers. We routinely notch ears to distinguish animals and further put unique marks on tails with a red marker to more easily and quickly distinguish animals.