eamag/protocols-with-steps huggingface dataset:
TITLE: Immunohistochemical classification of sensory and autonomic neurons projecting to the lower urinary tract in rats [keast-001]
AUTHORS: Janet Keast, Peregrine Osborne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This collection describes the procedures required to label, visualise, characterise and quantify neurons that innervate the lower urinary tract tissues of adult male and female Sprague-Dawley rats. This collection includes protocols for:</div><div class = "text-block">STAGE 1: Surgery to micro-inject fluorescent retrograde tracer dyes into one or more sites within the lower urinary tract</div><div class = "text-block">STAGE 2: Intracardiac perfusion with fixative to preserve neural tissues of interest</div><div class = "text-block">STAGE 3: Fluorescence immunohistochemistry of ganglion cryosections.</div></div>

[STEPS]
TITLE: Analysis of qualitative data, Statistical Analysis and Health Economic Analysis
AUTHORS: Kajiru Gad Kilonzo, Stefanie J. Krauth, Jo Halliday, Clive Kelly, Stefan Siebert, Gloria Temu, Christopher Bunn, Nateiya M Yongolo, Sally Wyke, Emma McIntosh, Blandina Mmbaga

[DESCRIPTION]
This protocol details analysis of qualitative data, statistical analysis and health economic analysis.

[STEPS]
SECTION: Analysis of qualitative data
1. REA data will be analysed using a constant-comparison method (34) to build up a preliminary classificatory scheme of the different kinds of joint pain reported in the community. Observation and documentary evidence will be used to situate the scheme.
SECTION: Analysis of qualitative data
2. Interview data will be analysed using thematic analysis to identify the major ways in which joint pain is experienced and managed, including sources of support and help.
SECTION: Statistical Analysis
3. Data from the community and hospital survey will be analysed using mixed-effect multivariable logistic and linear regression models with random effects for village, household, and observer to describe the prevalence and associated factors for MSK disorder among participants.
SECTION: Statistical Analysis
4. Stepwise selection, likelihood ratio test, and parsimony will be used for model refinement and optimisation.
SECTION: Health Economic Analysis
5. All health and social care resource use identified and measured in the health economic components of the surveys will be valued using available local/regional unit costs.
SECTION: Health Economic Analysis
6. Estimates of productivity loss (absenteeism and presenteeism) will be generated by attaching proxy values (e.g. average wage, by type of occupation, age and gender) to measured time off work, using the human capital approach.
SECTION: Health Economic Analysis
7. Preference based quality of life values from the prevailing Ugandan value set for the EQ-5D and CHU-9D will be attached to EQ 5D responses for all adults/children respectively.
SECTION: Health Economic Analysis
8. An econometric model based on the statistical relationships between quality of life and healthcare resource utilisation and socioeconomic characteristics will be built using standard regression analysis, accounting for clustering (i.e. by household/village) as well as specificities of health economics quality of life and cost data (non-normal, skewed distribution).
SECTION: Health Economic Analysis
9. The impact of MSK on catastrophic expenditures will be explored using principle components analysis, as well as calculating wealth asset scores.
SECTION: Health Economic Analysis
10. Results will be incorporated into a risk-based prediction algorithm to identify individuals and households at increased risk of poorer outcomes.
SECTION: Health Economic Analysis
11. Regression models will be run for predicting income/ asset levels and or Health related quality of life. The impact on the average utilisation of health services will be identified and differential effects with socioeconomic characteristics tested.
TITLE: Running Gels: Electrophoresis Gel Procedure
AUTHORS: Demian F Gomez, Andrew J. Johnson, You Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to run electrophoresis gels at the UF Forest Entomology Lab.</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: Co-immunoprecipitation using GFP-trap
AUTHORS: Dan Dou, Erika Holzbaur

[DESCRIPTION]
Here, we describe performing co-immunoprecipitation experiments in HEK293 cells using GFP-trap beads

[STEPS]
1. See "Protocol: HEK293 cell culture for co-immunoprecipitation experiments" for preceding culture and transfection. For the present study, HEK cells were transfected with EGFP-tagged bait proteins. Prey proteins were either endogenous or transfected with HA- or SNAP-tags. 

2. Prepare wash buffer:

 

 

3. Prepare lysis buffer. Lysis buffer composition differed for experiments needing lambda phosphatase treatment
3.1. Lysis buffer for non-lambda phosphatase experiment:
 

3.2. Lysis buffer compatible with lambda phosphatase experiment:

 


4. 24 hours after transfection, wash HEK cells twice in ice-cold PBS and lyse in appropriate lysis buffer. Use 600 µL lysis buffer per experimental condition (pooled across the three 10cm dishes).
5. Clarify lysates at 10 x g at 4 degrees C for 10 minutes
6. Wash 25 µL GFP-trap beads per experimental condition in 500 µL wash buffer, in low protein binding Eppendorf tubes under rotating agitation. 
 

7. Equilbrate beads in lysis buffer for 5 min at 4 degrees C under rotating agitation
9. Incubate beads with clarified lysate (or lambda phosphatase-treated lysate) for 1 hour at 4 degrees C under rotating agitation
8. For lambda phosphatase experiments: add 60 µL of 10 mM MnCl2 and 24 µL lambda phosphatase (2,400 units; New England BioLabs) for a final reaction volume of 600 µL per experimental condition. 
 

10. Wash beads three times for 5 min in wash buffer at 4 degrees C under rotating agitation
8.1. Incubate at 30 degrees C for 30 minutes
11. Resuspend beads in 60 µL denaturing buffer, and boil for 10 minutes to release bound protein
TITLE: Mitochondrial DNA base editing in HEK293T cells
AUTHORS: Nicole Lake, Kaiyue Ma, Justin Cohen, Monkol Lek

[DESCRIPTION]
This protocol is for the transfection of mitochondrial-targeted DddA-derived cytosine base editors (DdCBE), and their subsequent selection, in HEK293T cells. This uses a dual plasmid system, where a ‘left’ and ‘right’ DdCBE are needed for editing. Enrichment of cells with both DdCBE halves is achieved by separate drug selection for the left and right plasmids.

[STEPS]
SECTION: Plating of the HEK293T Cells
1. Plating of  

SECTION: Plating of the HEK293T Cells
1.1. The day before transfection, trypsinize and count the cells.
SECTION: Plating of the HEK293T Cells
1.2.  In a  , plate 150000 cells per well in 1 mL per well of complete HEK media (  with 10 % volume   without antibiotics). 
 

    
SECTION: Transfection of DdCBE Plasmids
2. Transfection of DdCBE Plasmids
SECTION: Transfection of DdCBE Plasmids
2.1. For each well of cells to be transfected, dilute 3 µL of   into 50 µL (total volume) of  and mix well. 

SECTION: Transfection of DdCBE Plasmids
2.2. In a separate tube, for each well of cells to be transfected, dilute 2 µg of each DdCBE plasmid   or  with   that had been modified to include PuroR marker (left and right, for total of 4 µg plasmid DNA (pDNA)) 
 
into 50 µL (total volume) of  . Then add 8 µL P3000 Reagent from   (a 2:1 ratio to DNA) directly to the diluted pDNA. Mix well.

SECTION: Transfection of DdCBE Plasmids
2.3. Add the diluted pDNA solution in P3000 reagent (from 2.2) to diluted   (from 2.1), mix, and incubate for15 min min at room temperature.

SECTION: Transfection of DdCBE Plasmids
2.4. Discard the old medium in the well. Add 1 mL complete HEK media (  with 10 % volume   without antibiotics) to each tube, mix well, and add to the corresponding well. Do this step well by well. Incubate the cells at 37 °C in a 5% CO2 tissue culture incubator.

SECTION: Plating of the HEK293T Cells
1.3. Wait for cells to attach   at 37 °C in a 5% CO2 tissue culture incubator.

SECTION: Selection of the Transfected Cells
3. Selection of the Transfected Cells
SECTION: Selection of the Transfected Cells
3.1. 1080 min hrs later, replace the medium with complete HEK media (  with 10 % volume   without antibiotics) containing up to   5 ug/mL of   and  1 ug/mL of   
SECTION: Selection of the Transfected Cells
3.2. Continue selection for 10-14 days and replace the medium every 2 days with complete HEK media containing   and  . Ensure drug selection is not finished until all the control untransfected cells are dead. Passage the cells to a larger well-size or flask if needed. 
SECTION: Selection of the Transfected Cells
3.3. Once selection is finished, maintain the cells in complete HEK media (  with 10 % volume   without antibiotics) for at least 2 days before performing any experiments.
TITLE: Qbiotix SARS-CoV-2 protocol
AUTHORS: vito 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Qbiotix has developed novel nucleic acid assays and sample preparation procedures to improve global microbial detection standards and rapidly/inexpensively test high volume of samples. Qbiotix aims to enable countries to test their entire populations, their food, their waters with millions of tests per day on a regular basis.</div><div class = "text-block">The assays are isothermal and homogeneous. Thermocycling is not required and amplification is non-enzymatic removing the need for complex instrumentation and reliance on variable quality enzymes. Critically we have removed the need for complex nucleic acid preparation, integrated the assay into the collection/transport solution and significantly reduced reliance on a complex supply chain. The assays remove a good proportion of the constraints of PCR which heretofore have inhibited the number of people being testing for SARS-CoV-2 in the US and globally. </div></div>

[STEPS]
?. 1- collect saliva vial  in a collection deviprovided by Qbiotix
1 mL
?. 2- open vial and scan barcode in a temperature laboratory with temperature between 18 and 25 degrees Celsius.
?. 3- dispense   sample to single well in plate with a standard pipette
250 µl
?. 4- dispense   of magnetic bead solution in well with a standard pipette in a temperature controlled room with temperature between 18 and 25 degrees Celsius.  The bead solution is supplied within a KIT provided by Qbiotix
250 µl
?. 5- incubate at controlled temperature on the plate reader
?. 6- place plate on a magnetic separator
?. 7- insert plate on analyzer (reading speed depends on chosen plate reader instrument). The plate reader analyzer needs to have with the ability to read fluorescence and control temperature
?. 6- read results
TITLE: Rehabilitation Protocol for Conservative Treatment of Knee Osteoarthritis 
AUTHORS: Luisa Mululo, Fabricio Loures

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Exercise guidelines for lower limbs for patients included in the multi-professional ambulatory for conservative treatment for knee osteoarthritis at Hospital Universitario Pedro Ernesto. </div></div>

[STEPS]
TITLE: Case - Plasma Insulin Measurement by ELISA
AUTHORS: Henri Brunengraber

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Mouse Insulin ELISA kit is used for the non radioactive quantification of insulin in mouse plasma.</div></div>

[STEPS]
?. Protocol:
1. Plasma or serum are isolated from mice fasted or after glucose injection. Heparin or EDTA coated tubes are acceptable for plasma fractionation.
2. Insulin is measured according to the manufacturer’s directions.
?. Manufacturer's Protocol below:MOUSE INSULIN ELISA KIT 96-Well Plate (Cat. #10-1247-01)
?. Principle of procedure (provided by manufacturer)Mercodia Mouse Insulin ELISA is a solid phase two-site enzyme immunoassay. It is based on the direct sandwich technique in which two monoclonal antibodies are directed against separate antigenic determinants on the insulin molecule. During incubation insulin in the sample reacts with peroxidase-conjugated anti-insulin antibodies and anti-insulin antibodies bound to microplate wells. A simple washing step removes unbound enzyme labeledantibody. The bound conjugate is detected by reaction with 3,3’,5,5’-tetramethylbenzidine. The reaction is stopped by adding acid to give a colorimetric endpoint that is read spectrophotometrically. This assay is a Sandwich ELISA based, sequentially, on:1) capture of insulin molecules from samples to the wells of a microtiter plate coated by pre-titered amount of a monoclonal mouse anti-rat insulin antibodies and the binding of biotinylated polyclonal antibodies to the captured insulin, 2) wash away of unbound materials from samples, 3) binding of horseradish peroxidase to the immobilized biotinylated antibodies, 4) wash away of free enzyme conjugates, and 5) quantification of immobilized antibody- enzyme conjugates by monitoring horseradish peroxidase activities in the presence of the substrate 3,3',5,5'-tetramethylbenzidine. The enzyme activity is measured spectrophotometrically by the increased absorbency at 450 nm, corrected from the absorbency at 590nm, after acidification of formed products. Since the increase in absorbency is directly proportional to the amount of captured insulin in the unknown sample, the latter can be derived by interpolation from a reference curve generated in the same assay with reference standards of known concentrations of rat insulin.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>GreenGlo ™, 20,000X in Water, is a non-carcinogenic and non-toxic alternative to Ethidium bromide used for the detection of nucleic acids in agarose gels. It is as sensitive as Ethidium bromide. There is no toxic DMSO as GreenGlo ™ is supplied in water. <br /> <br />GreenGlo ™ has fluorescence excitation maxima at 295 nm and 490 nm. The fluorescence emission maxima is similar to EtBr when bound to DNA – at 530 nm.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Calibration procedure for functionally aligning inertial sensors fixed to the shanks, thighs, trunk (e.g. sacrum, sternum), head, arms, and forearms / wrists. The movements were originally designed for analyzing skiing movements where the person is wearing ski boots. However, the same protocol can be applyied also barefoot or with regular shoes.</p>
<p> </p>
<p>Please refer to the protocol's guidelines for more information how to obtain a sample data set and the matlab code needed to process the functional calibration.</p>

[BEFORE_START]
<p>Place the inertial sensors on the shanks, thighs, and trunk (e.g. sacrum, sternum), head, arms and forearms / wrists. The exact sensor orientation does not matter, i.e. they don't need to be put in the exact same way for each new experiment. However, the sensors should be placed such as to reduce soft tissue artefacts. </p>
<p>For example, a good location for the shank sensors is the tibial plateau, about 10-15cm below the knee, slightly medially.</p>
<p>The arm sensors should not be placed on or too close to the biceps and the forearm sensor should be placed as close as possible to the wrist so that the axial rotation of the forearm can also be measured accurately.</p>

[GUIDELINES]
<p>This protocol is an extension of the protocol <a href="https://www.protocols.io/view/functional-calibration-for-trunk-and-lower-limb-fi-itrcem6" target="_blank">Functional calibration for trunk and lower limb fixed inertial sensors</a>. Most of the steps are identical to that protocol and the corresponding videos showing the movements from the front and side can be viewed there. This protocol adds one step, arm swing, that allows also to functionally calibrate upper-limb fixed inertial sensors.</p>
<ul>
<li>Before each movement, the person should remain motionless for 1-2 seconds to allow better data segmentation.</li>
<li>Trunk rotation: ski poles are only needed to ensure a better control of the rotation axis. The movement can also be performed with the hands held behind the head.</li>
<li>Hip ad/abduction: ski poles are only needed for balance. The movement can be performed without ski poles but requires quite good balance control abilities of the person.</li>
<li>Note: the order of the calibration movements is not important since data processing afterwards is independent of this order. However, it is important to once choose an order and then stick to it forever, to avoid confusion and mistakes.</li>
</ul>
<p> </p>
<p><em><strong>The code to analyze the data along with an example data set from an alpine skiing athlete is available on Code Ocean, under the DOI 10.24433/CO.23792aee-07c5-4cdc-bfe9-9e85fa1bf5d5 (<a href="https://codeocean.com/2017/10/16/inertial-sensor-functional-calibration-and-joint-drift-correction/code" target="_blank">https://codeocean.com/2017/10/16/inertial-sensor-functional-calibration-and-joint-drift-correction/code</a>).<br /></strong></em></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Human ovarian tissue fixation
AUTHORS: hannah.anvari, pooja.devrukhkar, francesca.e.duncan francesca.duncan

[DESCRIPTION]
Purpose: This protocol is intended for use in fixing human ovarian tissue in a research setting. This protocol supplements the steps detailed in the collection, processing, and long-term storage of human ovarian tissue as formalin-fixed paraffin-embedded (FFPE) samples in protocol: dx.doi.org/10.17504/protocols.io.e6nvwdk29lmk/v1.

[GUIDELINES]
Researchers working with human specimens will adhere to all safety and training protocols required by the institution (Northwestern Medicine/Northwestern University) including but not limited to:    


Biosafety Certification
Bloodborne Pathogens Certification
Working with Formaldehyde Certification
Collaborative Institutional Training Initiative (CITI program) certification

[STEPS]
SECTION: Ovarian tissue fixation for fixed wedges and ovarian explant pieces
1. Fill 1.5 mL micro centrifuge tube with 1 mL of Modified Davidson’s Solution (Cat. #64133- 50; Electron Microscopy Sciences, Hatfield, PA)
SECTION: Ovarian tissue fixation for fixed wedges and ovarian explant pieces
2. Add one ovary wedge or all tissue pieces (ovarian explant) from one experimental well per 1.5 mL micro centrifuge tube.
SECTION: Ovarian tissue fixation for fixed wedges and ovarian explant pieces
3. Allow all tubes to rock @ room temperature (RT) for 2-4 hrs.

SECTION: Ovarian tissue fixation for fixed wedges and ovarian explant pieces
4. Transfer tubes to cold room and rock at 4°C overnight (Fig. 1).
SECTION: Ovarian tissue fixation for fixed wedges and ovarian explant pieces
5.   

SECTION: Ovarian tissue fixation for fixed wedges and ovarian explant pieces
6. Wash in 1 mL 70% EtOH 3x10 minutes, rocking in between each, and then store in 70% EtOH @ 4°C until processing/paraffin embedding, no longer than 1 week.
SECTION: Ovarian tissue fixation for fixed wedges and ovarian explant pieces
7. Ovarian tissue wedges containing both cortex and medulla should be embedded so that the cortex and medulla will be sectioned in one plane (Fig. 1B). A representative H&E tissue section indicates the proper orientation (Fig. 1C).

SECTION: Ovarian tissue fixation for fixed wedges and ovarian explant pieces
8. Ovarian tissue pieces from explant culture should be embedded together, if cultured in the same well (Fig. 1D). While embedding try to ensure that tissues sit as evenly as possible, so that they are sectioned in the same plane.
TITLE: Library negative controls (LNCs) prepared on the Bravo NGS Workstation
AUTHORS: Anna Schmidt, Sarah Nagel, Matthias Meyer

[DESCRIPTION]
Protocol for the preparation of library negative controls (LNCs) in FluidX tubes using the Bravo NGS Workstation. LNCs are used as part of the library preparation workflow of the Ancient DNA Core Unit of the MPI-EVA.

[STEPS]
TITLE: Calcium chloride transformation buffer
AUTHORS: Andreas Sagen

[DESCRIPTION]
Calcium chloride (CaCl2) transformation is a laboratory technique in prokaryotic (bacterial) cell biology. The addition of calcium chloride to a cell suspension promotes the binding of plasmid DNA to lipopolysaccharides (LPS). Positively charged calcium ions attract both the negatively charged DNA backbone and the negatively charged groups in the LPS inner core. The plasmid DNA can then pass into the cell upon heat shock, where chilled cells (+4°C) are heated to a higher temperature (+42°C) for a short time.

[STEPS]
SECTION: 5 M Calcium chloride transformation buffer
1. Add 40 mL distilled water to a 50 mL tube
SECTION: 5 M Calcium chloride transformation buffer
2. Measure and add 36.7525 g Calcium chloride dihydrate

Materials:
 
SECTION: 5 M Calcium chloride transformation buffer
3. Adjust pH to pH 7.4 with concentrated sodium hydroxide or hydrogen chloride
SECTION: 5 M Calcium chloride transformation buffer
4. Add distilled water to 50 mL
SECTION: 5 M Calcium chloride transformation buffer
5. Filter sterilize with a 0.2 µm pore-size filter
TITLE: Spatial index of urban environment for health
AUTHORS: Mara Lucia Marques, Dalton Muller Pessoa Filho

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Purpose: </span><span>The objective was to design an urban index for the identification of urban environment propensity for physical activity (PA).</span></div><div class = "text-block"><span style = "font-weight:bold;">Methods</span></div><div class = "text-block"><span style = "font-weight:bold;">Geographic contextualization</span></div><div class = "text-block">Determine social and demographical features. For the present study, the municipality of Jataí is located in the southwest region of the state of Goiás, Brazil, has an area of 7,174.225 km², and an estimated population in 2017 of 98,128 people (IBGE, 2017). Its relief is flat and mildly undulating, with an altimetric variation between 500 and 1,100 m. Depicted localization area Figure.</div><div class = "text-block"><span style = "font-weight:bold;">Data sampling</span></div><div class = "text-block">Determine the local. For the present study, field data collection was carried out in urban public environments that showed the highest attendance rates of PA practitioners and with suitable structures such as walking/running tracks and outdoor gyms.</div><div class = "text-block">To identify the spatial urban health index (SUHI), data on demographic, environmental, and urban structure were collected. For the present study were consider number of residents (IBGE, 2011), parameters regarding road connectivity and land use and vegetation cover (SIEG, 2018), and the urban area slope parameter generated by the Shuttle Radar Topography Mission (MDE – SRTM), with a spatial resolution of 30 m (EMBRAPA, 1979).</div><div class = "text-block">Use GIS. For the presente study, a geographic database was prepared with GIS ArcGis 10.5.1 (ESRI v. 2018) for the composition of the physical and demographic analysis parameters and mapping of the SUHI by using the weighted multi-criteria overlay method.</div></div>

[STEPS]