eamag/protocols-with-steps huggingface dataset:
TITLE: UPitt TriState SenNet TMC Tissue Collection
AUTHORS: Marta Bueno, Lanping Guo, Ricardo Pineda, John Sembrat, koenigshoffm, Oliver Eickelberg

[DESCRIPTION]
This document outlines the tissue sampling of lung and heart specimens at the TriState SenNet TMC Biospecimen Core at the University of Pittsburgh, as part of the Cellular Senescence Network Program (SenNet).

[STEPS]
SECTION: Preparation
1. Remove specimen from shipping or storage container.
SECTION: Preparation
2. Place specimen inside the hood over dissection table and photograph prior to sampling.
SECTION: Preparation
3. Select the sites from which samples will be collected, annotate and mark (if serial sampling is done)
SECTION: Preparation
3.1. For lung:
Pleura (A)
Broncho-vascular bundle (B)
Parenchyma (C)
SECTION: Preparation
3.2. For heart:
Left Ventricle (D)
Right Ventricle (E)
Left Atrium (F)
Right Atrium (G)
SECTION: Flash freezing and storing
4. Cut each of the selected regions from the previous step into 9 small chunks. Chunks should be small enough to just cover the bottom of a microfuge tube (~5mm x 5mm x 5mm).
SECTION: Flash freezing and storing
5. Dab the small chunks on a sterile gauze pad and place each one into a labeled microfuge tube.
SECTION: Flash freezing and storing
6. Place 9 small chunks into the empty tubes to be snap frozen. 
Drop tubes into liquid Nitrogen. 
Leave for ~2-10 minutes. 
Remove and store in -80C freezer.
SECTION: Storing in formalin
7. Place one piece of the selected regions into a tubes containing 20x volume of 10% formalin (to be processed into FFPE blocks). 
Store in 4C fridge.

Note: Fixation occurs at a rate of 1 mm per hour. Over-fixation causes tight cross-linking and under-fixation can compromise the tissue.
SECTION: Storing for fresh processing
8. Place one piece of the selected regions into a tube with cold DMEM with anti/anti.
Store in 4C fridge. 

Process as soon as possible.
SECTION: Preparation
3.3. (OPTIONAL - If needed) 
Wash the full-thickness core in cold PBS in a petri dish.
SECTION: Storing in formalin
7.1. Make a notation of the time the sample went into formalin.
SECTION: Storing in formalin
7.2. Based on the size of the tissue, fix the required length of time (4 to 24 hours).
SECTION: Storing in formalin
7.3. After the fixation period, remove the cassette and place in 70% ETOH until scheduled processing.
TITLE: Determination of edits in CRISPR-edited cell lines by sequencing
AUTHORS: Thanh Ngoc Nguyen

[DESCRIPTION]
This protocol details the procedure of determination of edits in CRISPR-edited cell lines by sequencing.

[STEPS]
SECTION: Procedure
1. Harvest the CRISPR-edited cells that need to be sequenced and the control parental cells.
SECTION: Procedure
3. Amplify the region of interest (CRISPR-target region) via PCR using primers obtained when designing CRISPR construct.
 

SECTION: Procedure
4. Run a 1 % DNA agarose gel to check if the PCR has worked.
SECTION: Procedure
5. If PCR products are present, clean them up with Qiagen PCR cleanup kit.
SECTION: Procedure
6. Send the cleaned-up PCR products to sequencing service with a sequencing primer.
 

SECTION: Procedure
7. Analyze the sequencing data using this website https://ice.synthego.com/#/.
SECTION: Procedure
8. Sometimes, if the sequencing service provider(s) have trouble sequence the PCR products, it might be worth trying to clone these PCR products into a small non-expression plasmid such as pGEM4Z prior to sequencing:

SECTION: Procedure
8.1. Incorporate BamHI site (GCGCGGATCC; BamHI site is highlighted in grey, the rest is overhang) and HindIII site (GCGCAAGCTT; HindIII site is highlighted in green, the rest is overhang) into the primers mentioned in step 3.
SECTION: Procedure
8.2. Amplify the region of interest from genomic DNA isolated from the CRISPR-edited cells with these primers via PCR.
SECTION: Procedure
8.3. Cut the amplified PCR products and pGEM4Z with BamHI and HindIII.
SECTION: Procedure
8.4. Clean up the cut PCR products and pGEM4Z with Qiagen PCR cleanup kit.
SECTION: Procedure
8.5. Ligate the PCR products and pGEM4Z together using T4 DNA ligase.
SECTION: Procedure
8.6. Transform the ligated product mix into E. coli competent cells and plate on an Ampicillin agar plate.
SECTION: Procedure
8.7. Screen for colonies with pGEM4Z ligated with the PCR products.
SECTION: Procedure
8.8. Send them for sequencing with M13 forward or reverse primer.
SECTION: Procedure
8.9. Align the sequencing data with the reference sequence to determine the edits.
SECTION: Procedure
2. Isolate genomic DNA using according to manufacturer’s instructions.
TITLE: Starch Concentration Protocol
AUTHORS: Victor Rodriguez

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The aim of this protocol is to extract and analyze the starch content of a plant in order to gain data into its metabolism and photosynthetic properties. </div></div>

[STEPS]
?. [Pigment Extraction]
Weigh out  of plant sample to be tested and add it to a 200ml beaker.
0.25 g
?. [Pigment Extraction]
Add  of to the test tube.
20 ml
?. [Pigment Extraction]
Let the sample/acetone suspension sit in a 200 ml beaker filled with distilled water and heat water until the of starts to boil.
?. [Pigment Extraction]
Let test tube cool for  minutes to extract the pigments of the plant.
?. [Pigment Extraction]
Drain the and add  moreand put it back into the beaker heat water until the of starts to boil.
20 ml
?. [Pigment Extraction]
Let test tube cool for  minutes more to finish extracting the pigments.
?. [Pigment Extraction]
Drain the test tube and transfer the sample to  an autoclave for heating for  hour at  . This will loosen up the starch for extraction.
135 °C
?. [Pigment Extraction]
Remove the sample from the autoclave.
?. [Pigment Extraction]
Grind the sample with a mortar and pestle until a paste.
?. [Pigment Extraction]
Add   of distilled water to the pestle and wash around to dissolve the sample into a suspension and clean the pestle. Ensure all of the sample is in the suspension.
5 ml
?. [Pigment Extraction]
Funnel the suspension into a test tube and add an additional    of distilled water to the test tube using the same funnel as to wash out the funnel of stuck on sample particles.
5 ml
?. [Pigment Extraction]
Let stand for an hour covered.
?. [Pigment Extraction]
Add  of the suspension to a 3 ml vial and   of the suspension to another 3 ml vial. Mark the vials as needed for the protocol. Add   of distilled water to the vial with 1ml. This will serve as a 1/3 diluted sample in case the concentration is too high for the spectrometer to read the original sample.
3 ml
1 ml
2 ml
?. [Pigment Extraction]
To two labeled PCR tubes, add  from each vial and centrifuge for  at  .
200 µl
Centrifuge: 1200 34
?. [Pigment Extraction]
Take   from each PCR tube and transfer it to clean PCR tubes labeled. Ensure that the sample is removed from the top to prevent sucking up settled particulates.
100 µl
?. [Pigment Extraction]
Add    of distilled water to a PCR tube and calibrate the Spectrometer with it.
100 µl
?. [Pigment Extraction]
Insert each sample into the spectrometer.
?. [Pigment Extraction]
The Absorbance of the samples are to be taken at the 610nm wavelength and the log base 10 of the absorbance percentage is noted.
?. [Pigment Extraction]
Use the following function to determine the concentration...Cst = (A610*692.7g/mol*Vml)/(2594*Msm)With Cst being the starch concentration in mg/g, A610 is the absorbance at 610 nm, Vml is the volume in ml of the water added to the sample for extraction, and Msm is the mass in grams of the original sample taken.
TITLE: Olfactory Organoids
AUTHORS: Alp Ozgun, Priya Suman, Josée Coulombe, Julianna Tomlinson, Earl G. Brown, John M. Woulfe, Michael G. Schlossmacher

[DESCRIPTION]
This protocol enables obtaining 3D matrix-embedded olfactory organoids from KRT5+ olfactory horizontal basal cells. Resulting samples consist of hydrogel matrix domes with a volume of 40 µL containing hollow spheres of olfactory epithelium. The amounts were given per sample and should be calculated according to how many samples will be made.


[STEPS]
3. Lift olfactory basal cells from the culture surface by rinsing with dPBS and adding cold accutase. Incubate at 37 °C for up to 15 minutes.

4. Add HBC medium equal to the volume of accutase and transfer the cells into a falcon tube. Centrifuge at 350 x g for 4 minutes and discard the supernatant. Resuspend cells in 2 mL HBC medium and perform cell count.

2. Place 24-well plates into tissue culture incubator at 37 °C to equilibrate the temperature of the plastic. This step is important to prevent sinking of the cells to surface inside the matrix precursor. 

5.   Transfer 4x104 cells per sample into a new falcon tube and centrifuge at 350 rpm . Discard supernatant. Add 15 µL organoid initiation medium per sample and resuspend the cells. Perform the following steps as quickly as possible.

1. Thaw reduced growth factor matrix precursor aliquots at 4 °C overnight. 20 µL matrix precursor is required per sample. Geltrex and Matrigel were tested and shown to work equally well with this protocol. Keep these products at 4 °C or on ice throughout the protocol. 

6. Add 30 µL matrix precursor per sample to the cell suspension and briefly triturate to homogenize. Retrieve 24-well plates from the incubator and pipette 40 µL of the cell suspension into the center of each well, forming a liquid dome. Immediately return the well plates to the incubator. Aim to finish pipetting and return the plates to the incubator within 3-4 minutes after adding the matrix precursor to the cells.

7. Allow the matrix solutions to set in the incubator for 30 minutes. Confirm that the cells did not settle to the bottom using an inverted microscope. Gently fill the wells with 500 µL  organoid initiation medium by pointing the pipette tip to the well wall.

8. Return the well-plates to the incubator. This day is denoted as 0 days-in-vitro (0 DIV).
9. At the end of 4 DIV, carefully aspirate the organoid initiation medium and add 500 µL  maintenance medium. At this stage, cell clumps and hollowing spheres should be observed under the microscope. 

10. Over the following days, spheres should start to hollow out. At the end of Day 7, supplement the maintenance medium with 3 micromolar (µM)  retinoic acid. The sphere walls will then start to thicken and differentiate into pseudostratified architecture of the olfactory epithelium. The samples are ready for further testing by 20 DIV.
TITLE: Adapter ligation with AMII v2
AUTHORS: Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a subprotocol for performing adapter ligation with AMII</div></div>

[STEPS]
?. In a new Eppendorf tube set up the following AMII adapter ligation reaction.  AB1ComponentVolume2Barcoded amplicon pool30 µL3NEBNext Quick Ligation Reaction Buffer (5X)10 µL4Adapter Mix (AMII)5 µL5Quick T4 DNA Ligase5 µL6Total50 µL
1.5 µl
AB1ComponentVolume2Barcoded amplicon pool30 µL3NEBNext Quick Ligation Reaction Buffer (5X)10 µL4Adapter Mix (AMII)5 µL5Quick T4 DNA Ligase5 µL6Total50 µL
?. Incubate at room temperature for
?. Add (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting. Pulse centrifuge to collect all liquid at the bottom of the tube.
50 µl
Vortex SPRI beads thoroughly before use to ensure they are well resuspended, the solution should be a homogenous brown colour.
There will be some variation in clean-up efficiencies but expect to carry around 50% through this clean-up
?. Incubate for  at room temperature.
?. Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. Add  SFB and resuspend beads completely by pipette mixing. Pulse centrifuge to collect all liquid at the bottom of the tube. Remove supernatant and discard.
250 µl
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.
?. Repeat steps 13.6 to perform a second SFB wash.
?. Pulse centrifuge and remove any residual SFB. Add EB (ONT) and resuspend beads by pipette mixing.
15 µl
You do not need to allow to air dry with SFB washes.
?. Incubate at room temperature for .
?. Place on magnetic rack until clear. Transfer final library to a new 1.5mL Eppendorf tube.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol refers to LVL 1 cloning with the Marburg Collection

[GUIDELINES]
This protocol refers to LVL 1 cloning with the Marburg Collection

[STEPS]
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Reaction Setup on ice
?. 
SECTION: Thermocycling conditions
?. 
SECTION: Thermocycling conditions
?. 
SECTION: Thermocycling conditions
?. 
SECTION: Thermocycling conditions
?. 
SECTION: Transformation
?.
TITLE: Quantifying the LAMP1 positive puncta
AUTHORS: Narayana Yadavalli, Shawn M. Ferguson

[DESCRIPTION]
This is describes quantifying the LAMP1 positive puncta.

[STEPS]
SECTION: Quantifying the LAMP1 positive puncta
1. Select cells of interest with similar shapes and without saturation, and isolate single ROI.
SECTION: Quantifying the LAMP1 positive puncta
2. Apply auto threshold to total stacks of the images.
SECTION: Quantifying the LAMP1 positive puncta
3. Estimate the number of LAMP1 positive vesicles in each ROI by using “Analyze particle” in ImageJ/Fiji.
SECTION: Quantifying the LAMP1 positive puncta
4. Plot the results by calculating the mean number of LAMP1 positive puncta per each cell.
TITLE: Nucleoside analysis with liquid chromatography–tandem mass spectrometry (LC–MS/MS) 
AUTHORS: Peter Thuy-Boun

[DESCRIPTION]
This protocol details the detection of modified nucleosides using LC–MS/MS

[STEPS]
SECTION: Chromatography
1. Perform online separation of nucleoside mixtures using a liquid chromatography (LC) setup paired to a mass spectrometer.
 

SECTION: Mass spectrometry
2. Acquire data using a mass spectrometer capable of MS/MS experiments (we used a Thermo LTQ Orbitrap XL outfitted with an API source, below are recommended settings).

 

SECTION: Data processing
3. Convert Thermo .raw files obtained after tandem mass spectrometry analysis to .mgf format using MSConvert 3.0.22031 (a component of the Proteowizard open source mass spectrometry bioinformatics software package) under generic default presets for .mgf file extraction.
SECTION: Analyte identification
4. Use the Jupyter notebook linked below to identify candidate nucleosides based on .mgf files extracted during the previous step.
 

Our GitHub repository with "nucleoside finder" script is available here: https://github.com/Arcadia-Science/nucleoside-finder/tree/v1.0
SECTION: Mass spectrometry
2.1. Acquire data in positive mode with lock-mass enabled (targets: 391.284290, 413.266230).
SECTION: Mass spectrometry
2.2. Adjust MS1 scan settings to target ions in the 200–800 m/z range at 100,000 resolution selecting for charge states +1 and +2 for MS/MS.
SECTION: Mass spectrometry
2.3. Follow each MS1 scan with 7 data-dependent MS2 scans collected also at 100,000 resolution. Set ion isolation window at 4 m/z.
SECTION: Mass spectrometry
2.4. Activate ions by CID at NCE of 35% with a minimum signal threshold of 3e4.
SECTION: Mass spectrometry
2.5. Enable dynamic exclusion with a maximum repeat count of three times within 30 s with an exclusion duration of 30 s.
SECTION: Chromatography
1.1. Prepare 20 µL of nucleoside digest containing approximately 1 µg of nucleoside mixture. Inject 10 µL of digest per run.
SECTION: Chromatography
1.2. Separate nucleosides using a C18 column and binary solvent gradient of HPLC-grade water + 0.1% formic acid (A) and acetonitrile + 0.1% formic acid (B).
SECTION: Chromatography
1.4. Create a chromatography method using the following 30 min gradient as a guide:

0–0.5% B over 2 min 
0.5–30% B over 4 min 
30–95% B over 0.5 min 
Hold at 95% B for 4 min 
95–0.5% B over 0.5 min
Hold at 0.5% B over 19 min
 


SECTION: Chromatography
1.3. Set LC flow rate at 150 µL/min.
 

SECTION: Chromatography
1.5. Enable column heater at a constant 45 °C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>Modified from <span style="color: #222222; font-family: inherit; font-size: small;">Csaky</span><span style="color: #222222; font-family: inherit; font-size: small;"> </span><span style="color: #222222; font-family: inherit; font-size: small;">T. Z.</span><span style="color: #222222; font-family: 'open sans'; font-size: xx-small;"> </span><span style="color: #222222; font-family: 'open sans'; font-size: xx-small;">(</span><span style="color: #222222; font-family: inherit; font-size: xx-small;">1948</span><span style="color: #222222; font-family: 'open sans'; font-size: xx-small;">) </span><span style="color: #222222; font-family: Arial, sans-serif; font-size: small;">On the estimation of bound hydroxylamine in biological materials.  Acta Chem. Scand. <span style="font-family: inherit;"><strong>2</strong></span>, <span style="font-family: inherit;">250</span>–<span style="font-family: inherit;">454</span></span></p>
<p><span style="color: #222222; font-family: Arial, sans-serif; font-size: small;"><span style="font-family: inherit;">and A. H. Gillam, A. G. Lewis, and R. J. Andersen. Quantitative determination of hydroxamic acids Anal. Chem., 1981, 53 (6), pp 841–844 DOI: 10.1021/ac00229a023</span></span></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties
AUTHORS: Anne Zemella, Theresa Richter, Lena Thoring, Stefan Kubick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Fluorescent labeling of de novo synthesized proteins is in particular a valuable tool for functional and structural studies of membrane proteins. In this context, we present two methods for the site-specific fluorescent labeling of difficult-to-express membrane proteins in combination with cell-free protein synthesis. The cell-free protein synthesis system is based on Chinese Hamster Ovary Cells (CHO) since this system contains endogenous membrane structures derived from the endoplasmic reticulum. These so-called microsomes enable a direct integration of membrane proteins into a biological membrane. In this protocol the first part describes the fluorescent labeling by using a precharged tRNA, loaded with a fluorescent amino acid. The second part describes the preparation of a modified aminoacyl-tRNA-synthetase and a suppressor tRNA that are applied to the CHO cell-free system to enable the incorporation of a non-canonical amino acid. The reactive group of the non-canonical amino acid is further coupled to a fluorescent dye. Both methods utilize the amber stop codon suppression technology. The successful fluorescent labeling of the model G protein-coupled receptor adenosine A2A (Adora2a) is analyzed by in-gel-fluorescence, a reporter protein assay, and confocal laser scanning microscopy (CLSM). Moreover, a ligand-dependent conformational change of the fluorescently labeled Adora2a was analyzed by bioluminescence resonance energy transfer (BRET).</div></div>

[STEPS]
TITLE: The Role of protein modification patterns in cancer and diabetes
AUTHORS: Cathy Miller

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Post-translational modification</span></a><span> (PTM) refers to the covalent modification and enzymatic modification of a protein after biosynthesis. The modification can occur in the amino acid side chain and its C or N terminus. More function are introduced through modification of functional groups or introduction of new groups (such as phosphates, acetyl groups, etc.). Most proteins expressed by eukaryotes undergo a series of post-translational processing and modification to become the final complex function performer, so post-translational modification of proteins becomes an important aspect of proteomics research.</span></div></div>

[STEPS]
TITLE: BAM Alignment to CP018802  for Submission to GenBank
AUTHORS: Gregory Harhay

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Create BAM Alignment FIles including necessary tags that designate library info for submission into the Sequence Read Archive (SRA) at NCBI.  Parking the BAM file at the SRA is convenent way to provide alignments to reproduce results at </span><a href="https://dx.doi.org/10.17504/protocols.io.wemfbc6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Steps to Create FASTQ of CCS Overlapping Genomic SSR - CCS ROI</span></a></div></div>

[STEPS]
?. [Software Requirements]
or another read mapper such as BowTie2
?. [Download genome from GenBank]
https://www.ncbi.nlm.nih.gov/nuccore/CP018802.1
?. [Align reads in each sequencing run to reference (CP018802)]
Nine PacBio CCS libraries were mapped to CP018802L.17494L.17495L.17836L.18256_1L.18256_2L.18257L.19770L.19771L.19981Mapping Parameters: Geneious Assembler,  Medium Sensitiivity/Fast, iterate up to 5 times, Map multiple best matched randomly for 9 libraries
?. [Edit metadata in each SAM - NCBI requires library identifier  metadata in read groups (@R)]
Edit each SAM with SAM tools to append library as read group (RG) ID and append sample RG tag to each of the  SAM alignments generated
 for each SAM, need to remove original @RG header line,  for example change @HD    VN:1.0    SO:unsorted @SQ    SN:CP018802    LN:2110642 @RG    ID:Unpaired_reads_assembled_against_CP018802    SM:L.17495_GeneMap_CP018802 @RG    ID:LB:17495    SM:63250 to  @HD    VN:1.0    SO:unsorted @SQ    SN:CP018802    LN:2110642 @RG    ID:LB:17495    SM:63250 and save file
?. [Merge SAM files into a single BAM]
?. [Upload to SRA with appropriate metadata]
BAM file available at  https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?run=SRR8080935 , the download BAM file is named SRR8080935_CP018802.bam
TITLE: High-Throughput Multiplex Live-Cell Imaging of iPSC-derived neurons
AUTHORS: james.evans

[DESCRIPTION]
Live-cell high-throughput imaging of multiplexed dyes to reveal organelle dynamics (nucleus, mitochondria and lysosomes) in live iPSC-derived neurons. 

[STEPS]
1. Live-cell multiplexed imaging was performed in 96-well plates (PerkinElmer). Cortical neurons were plated at a density of 20,000 - 40,000 cells/well after at least 50 days of neural induction. Neurons were then maintained until imaging (>70 days) with twice weekly feeding.
2. Cells were then washed with Hank’s balanced salt solution (HBSS; Invitrogen)
3. Cells were then incubated with 10 μM Hoechst (62249, ThermoFisher Scientific), 25 nM TMRM (T668, Thermo Fisher Scientific) and 250 nM LysoTracker Deep Red (L23492, Thermo Fisher Scientific); 500 nM SYTOX green (S7020, Thermo Fisher Scientific) in HBSS for 30 minutes at room temperature.
4. Live-cell images were acquired using an Opera Phenix High-Content Screening System (PerkinElmer). Hoechst was imaged using the 405 nm laser, SYTOX green labelling was imaged using the 488 nm laser, TMRM was imaged using the 516 nm laser, and LysoTracker was imaged using the 647 nm laser. Live-cell imaging was performed in HBSS. ~20 fields of view were taken per well with each field taken at 5 focal planes in the z-axis.
TITLE: PEGylation Analysis and Method Verification
AUTHORS: BOC       Sciences

[DESCRIPTION]
BOC Sciences provides comprehensive PEGylation analysis services in terms of qualitative assessment, quantitative assessment and in vivo & in vitro characterization.
After PEGylation, conjugates will show changes in physiochemical properties, such as surface charge, hydrodynamic diameter, hydrophilicity and protein binding capability. By monitoring changes in these physiochemical properties, PEGylation can be confirmed.


[STEPS]