eamag/protocols-with-steps huggingface dataset:
TITLE: LDW_BDC Muscle scRNAseq
AUTHORS: Lauren D Walter, Benjamin D Cosgrove

[DESCRIPTION]
single-cell RNA sequencing of muscle

[STEPS]
SECTION: Skeletal muscle injury and collection
1. In a given mouse, both tibialis anterior (TA) muscles were injected with 10 µl of notexin (10 µg/ml; Latoxan, France).
13. 
SECTION: Skeletal muscle injury and collection
13.      The mice were sacrificed, and TA muscles were collected at 0, 1, 2, 3.5, 5, and 7 days post-injury. Each TA was processed independently to generate single cell suspensions.
SECTION: Skeletal muscle injury and collection
2. At various days post injury, the mouse was sacrificed and the TAs were collected.  Each TA was proccessed independently to generate single cell suspensions.
SECTION: Generate single-cell suspensions
3. Muscles were enzymatically digested with 8 mg/ml Collagenase D (Roche, Basel, Switzerland) and 10 U/ml Dispase II (Roche, Basel, Switzerland) and then manually dissociated to generate cell suspensions. 
SECTION: Generate single-cell suspensions
4. Myofiber debris was removed by filtering the cell suspensions through a 100 µm and then a 40 µm filter (Corning Cellgro # 431752 and # 431750). 
SECTION: Generate single-cell suspensions
5. After filtration, erythrocytes were removed by incubating the cell suspension in erythrocyte lysis buffer (IBI Scientific # 89135-030).
SECTION: Generate single-cell suspensions
6. After digestion, the single-cell suspensions were washed and resuspended in 0.04% BSA in PBS at a concentration of 106 cells/ml. A hemocytometer was used to manually count the cells to determine the concentration of the suspension. 
SECTION: Single-cell RNA library preparation and sequencing
7. Single-cell RNA-sequencing libraries were prepared using the Chromium Single Cell 3’ reagent kit v3 (10x Genomics, Pleasanton, CA) following the manufacturer’s protocol. 
SECTION: Single-cell RNA library preparation and sequencing
8. Cells were diluted into the Chromium Single Cell A Chip to yield a recovery of 6,000 single-cell transcriptomes with <5% doublet rate. 
SECTION: Single-cell RNA library preparation and sequencing
9. Libraries were sequenced on the NextSeq 500 (Illumina, San Diego, CA).
TITLE: U Mass - Alkaline Phosphatase
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">
This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of alkaline phosphatase reflect liver function.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry
analyzer.
?. Select Alkaline Phosphatase test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Phosphate Buffer
AUTHORS: Neilier Junior

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A buffer solution has the function of resisting changes in pH even when adding powerful acids or bases. However, in the physiological environment the buffered system also provides cofactors for enzymatic reactions, critical salts and even essential nutrients for cells and tissues. Therefore, when trying to reproduce biological conditions in vitro, we must make the appropriate choice of the buffer. After all, it will provide the appropriate medium in which reactions will occur.</div></div>

[STEPS]
?. [Phosphate Buffer]
Mix sodium phosphate monobasic and dibasic solutions in the proportions indicated.  ABCDEFGHIJKL1mL of Sodium phosphate, Monobasic92.081.573.562.551 .039.028.019.013.08.55.32mL of Sodium phosphate, Dibasic8.018.526.537.549.061 .072.081.087.091.594.73pH5.86.26.46.66.87.07.27.47.67.88.0
pH range:   to  
(a) 0.1 M Sodium phosphate monobasic; 13.8 g L-1 (monohydrate, M.W. 138.0 g mol-1)(b) 0.1 M Sodium phosphate dibasic; 26.8 g L-1 (heptahydrate, M.W. 268.0 g mol-1)
ABCDEFGHIJKL1mL of Sodium phosphate, Monobasic92.081.573.562.551 .039.028.019.013.08.55.32mL of Sodium phosphate, Dibasic8.018.526.537.549.061 .072.081.087.091.594.73pH5.86.26.46.66.87.07.27.47.67.88.0
?. [Phosphate Buffer]
Adjust the final volume to  with deionized water.
200 ml
?. [Phosphate Buffer]
Adjust the final pH using a sensitive pH meter.
TITLE: Plasmid transduction using competent cell
AUTHORS: An.Huang

[DESCRIPTION]
Plasmid can be transduced into bacteria at competent state using heat shock. This protocol helps transduce plasmid into competent cells.

[STEPS]
1. Take competent cell out from -80℃ fridge and thaw on ice.
2. When the cells are completely thawed, pipette 2 µL plasmid DNA solution into 100 µL competent cell.
Put the cell in ice for 30 min

3. Conduct heat shock on the competent cell by placing the cell in 42 °C water bath for 90 s.
Put the cells back into ice for 2 min

4. Add 900 µL LB broth medium into competent cell mixture. Shake at   for 45 min

5. Centrifuge at 6000 rpm, 5 min.
 

6. Discard 900 µL supernatant and resuspend the pellet in the rest 100 µL supernatant.

7. Spread the cells onto LB agar plates.
 

8. Place the plate with lid on upside for 60 min.

9. Invert the plate and culture at 37 °C in a biomedical incubator overnight.
TITLE: Expression and purification Twin-STREP-FLAG tagged ATG13:ATG101 constructs
AUTHORS: Adam    Yokom, Xuefeng  Ren

[DESCRIPTION]
Expression and purification Twin-STREP-FLAG tagged ATG13:ATG101 constructs

[STEPS]
SECTION: Expression
1. Transfect HEK GNTI cells at concentration of 2 × 106 cells/ml
   
SECTION: Purification
8. Resuspended pellet in lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol) with 1% Triton X-100 and protease inhibitor cocktail (Thermo Scientific, Waltham, MA) for 15 min
SECTION: Expression
2. Dilute PEI with Warm Hybridoma-SFM(1X)
SECTION: Purification
9. Clarify lysate for 17000 rpm, 30 min, 4 °C

SECTION: Purification
11. Rock supernatant with equilibrated resin for 60 min at 4 °C
    Use glutathione resin for GST tagged proteins
    Use amylose resin for MBP tagged proteins
    Use Strep-Tactin Sepharose resin for Strep tagged proteins
SECTION: Purification
10. Wash strep-tactin resin (IBA Lifesciences, Germany) into lysis buffer (without Triton). Load clarified lysate onto resin
SECTION: Purification
12. Wash with 5CV lysis buffer (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA, 10% Glycerol)
SECTION: Purification
14. Concentrate elution and inject onto pre-equilibrated S200 10/30 column (25 mM HEPES pH 7.5, 200 mM NaCl, 2 mM MgCl2, 1 mM TCEP, 5 mM EDTA)
SECTION: Purification
13. Elute with lysis buffer plus 4 mM desthiobiotin for STREP resin
SECTION: Purification
15. Pool peak fractions, concentrate, snap freeze, and store at -80C 
SECTION: Expression
3. In a separate tube, dilute DNA with Hybridoma-SFM(1X)
SECTION: Expression
4. Add PEI to DNA dilution. Incubate mixture for 30 min  at 37 °C 

SECTION: Expression
5. Add mixture to cells. Let cells grow for 2880 min 

SECTION: Expression
6. Harvest Cells 500 rpm, 10 min, 4 °C 

SECTION: Expression
7. Wash pellet with cold PBS. Store pellet at -80C until purification.
TITLE: Whole Cell Patch Clamp of Dispersed Human Islet Cells
AUTHORS: Xiaoqing  Dai, Austin Bautista, Patrick E Macdonald

[DESCRIPTION]
Cells use exocytosis to secrete a wide variety of molecules, including proteins, hormones, and neurotransmitters. Exocytosis can be monitored at the single-cell level by using patch-clamp electrophysiology to measure changes in membrane capacitance as vesicles fuse with the cell membrane and release their content. Dispersion of pancreatic islets into single cells allows for individual characterization of electrophysiological characteristics and allows for collection of cellular content for recovery of full-length transcriptomes by use of Smart-seq2.

Described in this protocol is the dispersion of pancreatic islets into single cells followed by whole-cell patch clamp electrophysiology which includes parameters representing cell size, exocytosis, sodium channel currents, and calcium channel currents. Cells are then collected individually after recording to be processed for single-cell RNA sequencing.


 



[GUIDELINES]

References

 


[STEPS]
SECTION: Procedure
1. On the day receiving the shipped human pancreatic islets, hand-picked islets are dissociated to single cells using StemPro accutase (Gibco/Fisher, A11105-01). Plate cells in   cell culture dishes, and culture in DMEM with 5.5 mM glucose, 10% FBS, and 100 U/mL penicillin/ streptomycin for 1-4 days.

SECTION: Procedure
2. Start patch clamping single cells after one overnight incubation, and continue patching cells for up to 4 days. Electrical activities are measured by using a pipette coated with sylgard (3 ~ 5 MΩ) in a heated chamber (32–35°C). Quality control is assessed by the stability of seal (>10 GΩ) and access resistance (<15 MΩ).
SECTION: Procedure
3. Perform electrical activity measurements in 1 minute from “break in” of cell membrane; measurement protocols include (in order): exocytosis, voltage-gated Na and Ca channel currents activated at -10 mV, and -120 mV, voltage-gated Na and Ca channel currents activated from -60 to +30 mV, steady-state inactivation of voltage-gated Na channel currents, reversal potential, hyperpolarization-activated non-selective cation currents activated at -140 mV.
SECTION: Procedure
4. When finishing all the measurements, use another big-tip pipette (0.2 ~ 0.5 MΩ) prefilled with 0.5 mL  lysis mix, suck the cell into the pipette, and transfer it into a 0.2 mL PCR tube prefilled with 4 mL lysis mix.

SECTION: Procedure
5. Save cells in -80 °C  freezer before shipping out for sequencing.

SECTION: Data Analysis
6. Using the software of Fitmaster (HEKA Instruments Inc, Germany), analysis is performed on the level of recording traces.
TITLE: Immunostaining of Human Frontal Cortex Sections
AUTHORS: Shiyi Wang

[DESCRIPTION]
Immunostaining of Human Frontal Cortex Sections

[STEPS]
1. **Tissue Acquisition**
1.1. - Obtain 40 μm thick floating sections of human frontal cortex from Banner Sun Health Research Institute in Sun City, Arizona.
1.2. - Use sections from 4 control subjects and 3 LRRK2 G2019S mutation carrier subjects.
2. **Subject Information**
2.1. - Verify that control subjects had no history of dementia, neurological, or psychiatric disorders at the time of death (Refer to Supplemental Table 1).
2.2. - Ensure that informed and written consent was obtained from all donors.
3. **Preparation of Sections**
3.1. - Wash sections in 1x TBS containing 0.3% Triton X-100 (TBST) to remove any residual fixative and debris.
4. **Blocking**
4.1. - Block non-specific binding by incubating sections in 3% normal goat serum (NGS) diluted in TBST for blocking, typically for 1 hour at room temperature.
5. **Primary Antibody Incubation**
5.1. - Incubate sections overnight at 4°C with gentle shaking in primary antibodies diluted in blocking buffer: GFAP (chicken, 1:250; AB5541, Millipore Sigma), Phospho-ERM (Rabbit, 1:250; #3726, Cell Signaling)
6. **Washing**
6.1. - Wash sections thoroughly with TBST to remove unbound primary antibodies.
7. **Secondary Antibody Incubation**
7.1. - Incubate sections with Alexa Fluor conjugated secondary antibodies (diluted 1:200 in TBST) for 2-3 hours at room temperature in the dark.
8. **Final Washing**
8.1. - Wash sections again with TBST to remove unbound secondary antibodies.
9. **Mounting**
9.1. - Mount sections onto glass slides using a homemade mounting media composed of: 90% Glycerol, 20 mM Tris pH 8.0, 0.5% n-Propyl gallate
10. **Sealing**
10.1. - Seal coverslips with nail polish to prevent drying and movement during imaging.
TITLE: Tail Suspension Test
AUTHORS: Marina Lorente Picón, Núria Peñuelas, Ariadna Laguna, Marta Gonzalez-Sepulveda, Miquel Vila

[DESCRIPTION]
Tail Suspension Test for mice

[STEPS]
1. Suspend animals by their tails with tape in a suspension bar.
2. To avoid the tail climbing behavior, pass a 2cm methacrylate tube through the tail before suspending the animal. 
3. Quantify the escape-oriented behaviors (i.e. fore and hind limbs movement) during six minutes. Vocalizations were registered during this period with a yes/no score.
4. Calculate the total immobilization time (s) as the sum of all the time the animal was not performing any escape-oriented behavior.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Here we describe two methods for assessing the ability of one bacteria to inhibit the growth of another through the production of antimicrobial compounds. In the first assay, the double agar layer method, only the secreted substances but not the candidate antagonist bacteria interact with the sensitive microorganisms. In the second assay, the simultaneous inhibition method, both microorganisms are grown together allowing the competitive exclusion.</p>

[STEPS]
TITLE: Conscious Cystometry Bladder Function Testing
AUTHORS: Firouz Daneshgari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Cystometry is a test of bladder function in which pressure and volume of fluid in the bladder is measured during filling, storage, and voiding. A cystometry study is performed to diagnose problems with urination, including incontinence and urinary retention.</div><div class = "text-block"><span style = "font-weight:bold;font-style:italic;">This test should be done on the mice two days after SPT Implantation.</span></div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bib64kcn.jpg" /></div><div class = "text-block"><span style = "font-weight:bold;">Reference </span></div><div class = "text-block">1. Cannon TW, Damaser MS: Effects of anesthesia on cystometry and leak point pressure of the female rat. Life Sci 69:1193-1202, 2001</div></div>

[STEPS]
?. Use scissors to cut the sealed tube located between the animal’s ears.
?. Attach the end of the tube not connected to the metabolic cage swivel device to the tube between the animal’s ears.
?. Start the saline syringed filled pump (Kent Scientific Corporation, Torrington, CT, USA) to flush debris within the animal’s tube for about 2 minutes.
?. Using the preset pump configurations (3 ml/hour) fill the bladder via a stopcock with room temperature saline and record bladder pressure by using a pressure transducer connected to an amplifier (BP-100, CB Sciences, USA) for a period of 2-5 hours.
?. Collect urine in a beaker mounted on a force transducer (FT-03 D, Grass Instrument Co, Quincy Massachusetts) placed beneath each cage.
?. The pressure and force transducers should be connected to a multi-port controller with software (Polyview, Grass Instrument Co, Quincy Massachusetts) to record data through the computer.
TITLE: Vandy - Mouse Echocardiography
AUTHORS: Chee Lim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Transthoracic mouse echocardiography is used to provide noninvasive imaging of the heart and allows for quantification of myocardialwall and chamber dimensions and systolic and diastolic performance.</div></div>

[STEPS]
?. The chest hair is first shaved and a topical delipatory agent (e.g. Nair) is used to remove any remaining body hair.
?. The conscious  mouse  is  held in the prone position, decreasing vagal reflexes  and associated abnormalities of heartrate or AV conduction. If  anesthesia  is  required, 3% isoflurane is  used  for  sedation  and  the  mouse  is  placed  in  a supine  position  on  a  heated  platform  (to  maintain  body  temperature)  with  embedded  ECG leads  (Visual Sonics). A nose cone is placed over the snout and isoflurane (1%) is delivered to maintain sedation throughout the procedure.
?. Ultrasound  coupling  gel  heated  to  34C  is  applied to  the  chest  area  and  a  linear  array transducer (18-23 MHz) is positioned to obtain two-dimensional B-mode parasternal long and short axis views at the mid-ventricular level(Vevo 2100, VisualSonics).
?. One-dimensional  M-mode  images  are  obtained  for  measurement  in  the  short  axis  view  to obtain cardiac wall and chamber dimensions.
?. Digital  images  are  permanently  archived.  Depending  on  the  type  of  measurement,  the imaging procedure can last from one to several minutes.
?. Left ventricular (LV)  chamber size and  wall thickness are  measured  off-line  in the M-mode from  at  least  three  consecutive  beats  and  averaged.  LV  wall  thickness:  intraventricular septum  (IVS)  and  posterior  wall  (PW)  at  systole  and  diastole;  and  LV  internal  dimensions (LVID) during systole and diastole are measured. LV percent fractional shortening (FS) and ejection fraction (EF) are calculated from the M-mode measurements.
TITLE: Fluo-3-AM Assay
AUTHORS: Thermo Fisher

[DESCRIPTION]
determine the intracellular Ca2+ concentration of your cells via a fluorescence marker

[STEPS]
1. dilute the DMSO stock solution (1-5mM) to 1-5μM in the buffered physiological medium of choice
2. Pluronic F-127 may be added to a concentration of 0.02 % volume 

4. incubate an aliquot of the cells with Fluo-3-AM for  60 min at 20 °C -37 °C 
5. wash the cells in indicator-free medium and incubate the cells for another 30 min in dye-free medium

3. mix in an aliquot of Fluo-3-AM stock solution to a concentration of 4 micromolar (µM) 

6. measure fluorescence at Ex/Em = 506/526 with a microplate reader
TITLE: Modified protocol for URA3 counter-selection at highly expressed regions of Saccharomyces cerevisiae
AUTHORS: Lenny Teytelman, Jasper Rine

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a modified protocol for </span><span style = "font-style:italic;">URA3 </span><span>counter-selection in </span><span style = "font-style:italic;">S. cerevisiae </span><span>and similar yeasts. It is identical to the standard counter-selection with two transformations, with the exception of the replica-plating step.</span></div><div class = "text-block"><span>This protocol is appropriate for genomic loci where </span><span style = "font-style:italic;">URA3 </span><span>is over-expressed and the Ura3 protein is likely to be present in the colonies and cells at higher toxic levels. Because this method required five times as many plates for the replica-plating, it is more wasteful and should only be used when necessary.</span></div></div>

[STEPS]
?. [Transformation and selection]
Perform the standard LiAc transformation to replace the locus of interest with the URA3 cassette.
?. [Transformation and selection]
Grow on CSM-Ura3 plates and confirm that the integration is correct by sequencing.
?. [Counter-selection against URA3]
Perform the standard LiAc transformation to replace the URA3 cassette with the target sequence.
?. [Counter-selection against URA3]
After the heat-shock, plate the cells onto YPD-plates, for    growth.
?. [Counter-selection against URA3]
Press the YPD plate onto the velvet on the replica-plating block.
?. [Counter-selection against URA3]
[Mock replica-plating] Press a new YPD plate against the velvet and discard the plate.
?. [Counter-selection against URA3]
[Mock replica-plating] Repeat Step6 another 4 times to slice through the layers with excess Ura3.
?. [Counter-selection against URA3]
[Replica-plating] After the five mock-YPD replicas, press a single 5-FOA plate against the same velvet.
?. [Counter-selection against URA3]
Grow the colonies and screen for the correct integration using sequencing.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
Prepare lysis buffer as described in guidelines.

[GUIDELINES]
2.0 mL Lysis Buffer is added to Sterivex immediately after filtration.&nbsp;Filters are stored at -20&deg;C or -80&deg;C.<br /><br />Lysis Buffer is: &nbsp;<br /><br />
<table>
<tbody>
<tr>
<td>Final Concentration</td>
<td>For 20 ml:</td>
</tr>
<tr>
<td>40 mM EDTA</td>
<td>1.6 ml of 0.5 M EDTA</td>
</tr>
<tr>
<td>50 mM Tris (pH 8.3)</td>
<td>1.0 of 1 M Tris (pH 8.3)</td>
</tr>
<tr>
<td>0.73 M Sucrose</td>
<td>5.13g sucrose</td>
</tr>
</tbody>
</table>
<br />* Note: Must use Phenol:Chloroform:IAA (25:24:1) pH 8.0! Add buffer supplied, shake well, allow phases to separate and check pH of buffer using pH paper. Add hydroxyquinoline to a final concentration of 0.1% (100 mg hydroxyquinoline per 100ml phenol:chloroform:IAA).

[STEPS]
?. 
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TITLE: Visium Direct Mount FFPE -- University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
The Visium Spatial Gene Expression for FFPE is designed to measure mRNA in tissue sections derived
from formalin fixed & paraffin embedded (FFPE) tissue samples and requires a Visium Spatial slide
with intact tissue sections as input. 

Tissue sections are processed with Visium Spatial Gene Expression assay performed by the University of Minnesota Imaging Center and sequenced by the University of Minnesota Genomics Center.



[STEPS]
SECTION: Deparaffinization, H&amp;E Staining, Imaging &amp; Decrosslinking
1.   

SECTION: Library Preparation &amp; Sequencing
2.   

 

SECTION: FASTQ Generation
3. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq software version 2.20.0
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol for RNA Purification form Plasma or Serum, using the ISOLATE II Biofluids RNA Kit. This protocol includes the lysate preparation procedure.</p>

[BEFORE_START]
<div class="page" title="Page 17">
<div class="section">
<div class="layoutArea">
<div class="column">
<p> </p>
</div>
</div>
</div>
</div>

[GUIDELINES]
<div class="page" title="Page 17">
<div class="section">
<div class="layoutArea">
<div class="column">
<p>Before you start:</p>
<ul>
<li>
<p>Plasma or serum of all human and animal subjects is considered potentially infectious. All</p>
<p>necessary precautions recommended by the appropriate authorities in the country of use</p>
<p>should be taken when working with plasma or serum.</p>
</li>
<li>
<p>We recommend the use of this kit to isolate RNA from plasma or serum prepared by a</p>
<p>standard protocol from non-coagulated, fresh whole blood using EDTA or sodium citrate as</p>
<p>the anti-coagulant.</p>
</li>
<li>
<p>Plasma prepared from fresh blood using heparin as an anti-coagulant is not suitable for use</p>
<p>with this protocol.</p>
</li>
<li>
<p>Due to the relatively low DNA content in plasma, the Genomic DNA Removal Column is not</p>
<p>necessary for this protocol.</p>
</li>
<li>
<p>It is recommended that no more than 200 μL of plasma or serum is used in order to prevent</p>
<p>clogging of the column.</p>
</li>
<li>
<p>Yields of RNA from plasma and serum is highly variable. In general, the expected yield</p>
<p>could vary from 1 to 100 ng per 100 μL plasma or serum used. In addition, the expected A<sub>260</sub>/A<sub>280</sub> ratio as well as the A<sub>260</sub>/A<sub>230</sub> ratio will be lower (&lt;1.8) than the normal acceptable range from other cells or tissues. Nonetheless, these isolated RNA can be effectively used in different downstream applications such as real-time PCR or microarrays.</p>
</li>
<li>
<p>Avoid multiple freeze-thaw cycles of the plasma or serum sample. Aliquot out the appropriate volume for usage prior to freezing.</p>
</li>
<li>
<p>It is important to work quickly during this procedure.</p>
</li>
</ul>
</div>
</div>
</div>
</div>
<p>Please review the Guidelines under <a href="https://www.protocols.io/view/genomic-dna-removal-and-total-rna-purification-fro-f48bqzw?guidelines" target="_blank">Genomic DNA removal and total RNA purification from all types of lysate</a> for other important details.</p>

[STEPS]
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