eamag/protocols-with-steps huggingface dataset:
TITLE: Automatic Deposition of DAN Matrix using a TM Sprayer for MALDI Analysis of Lipids
AUTHORS: Elizabeth Neumann, Carrie  Romer, Jamie Allen, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block">To describe the procedure for spraying tissue sections with DAN for imaging lipids.</div><div class = "text-block">Expected Outcome:</div><div class = "text-block">Slides should be coated with DAN.  Tissue sections should be imaged within 24 hours of matrix application.</div></div>

[STEPS]
?. [Autofluorescence Scan]
Remove slides from freezer and place in desiccator for  .
?. [Autofluorescence Scan]
Scan for autofluorescence on Zeiss Axio Scanner.
?. [TM Sprayer Setup]
Change nitrogen to 6 psi and turn on sprayer.
?. [TM Sprayer Setup]
Open software and set nozzle temperature to
40 °C
?. [TM Sprayer Setup]
Change LC solvent to A1 to 100% acetonitrile at 0.05 mL/min.
?. [TM Sprayer Setup]
“Load” sample loop with   acetonitrile.
5 mL
?. [TM Sprayer Setup]
Switch sprayer to “Inject” and spray for 2 minutes.
?. [TM Sprayer Setup]
“Load”   of matrix solution into sprayer loop.
6 mL
?. [TM Sprayer Setup]
Switch sprayer to “Inject” and spray for ~1 minute.  Use slide to check that matrix is flowing.
?. [TM Sprayer Setup]
Tape slide(s) onto  heated block on top of stage and adjust software scanning area.
75 °C
?. [TM Sprayer Setup]
Set method in software:    1350 mm/min nozzle velocity     1.5 mm track spacing    0.05 mL/min flow rate    CC Pattern    2 L/min flow rate    5 passes    40mm nozzle height    No drying time
?. [TM Sprayer Setup]
Save method and make sure it is highlighted.
?. [TM Sprayer Setup]
Under Cycle, click “Start,” then click “Continue.”
?. [TM Sprayer Setup]
When finished, remove slide and place in slide box.
?. [Cleanup]
Set TM sprayer temperature to  .
30 °C
?. [Cleanup]
Switch A1 solvent line to 100% ACN and set flow rate to 1 mL/min.
?. [Cleanup]
“Load” loop with   acetonitrile and “Inject” loop for  .
20 mL
?. [Cleanup]
Spray tip of nozzle with 5 mL of MEOH.
?. [Cleanup]
Log the pressure at your flow rate post cleaning. It must be within 3-4 psi of the starting pressure; if it is not, the cleaning procedure must be repeated.
?. [Cleanup]
Change the HLPC flow rate to 0.05 mL/min.
?. [Cleanup]
Spray nozzle with methanol.
?. [Cleanup]
Remove the foil and wipe stage down with methanol.
?. [Cleanup]
Replace wypall and bench diapers.
?. [Cleanup]
Place all syringes in the biohazard container and empty the solvent waste.
?. [Cleanup]
Shut down the software and turn off the TM Sprayer.
?. [Cleanup]
Shut the nitrogen off.
TITLE: Neuromelanin Processing and Image Acquisition
AUTHORS: Anastasia Filimontseva, YuHong Fu, Glenda Halliday

[DESCRIPTION]
Protocol for preparing post-mortem tissue for intracellular NM quantification.

[STEPS]
SECTION: Tissue Processing
1. Formalin-fixed paraffin-embedded tissue blocks from the pontine and midbrain regions were sectioned at 6µm, collected onto adhesive microscope slides and allowed to dry in the oven at 37°C for 48 hours.
SECTION: Tissue Processing
2. Slides were incubated in the oven at 60°C for 1 hour to melt the paraffin.
SECTION: Tissue Processing
3. To remove the paraffin, sections were submerged in HistoChoice Clearing Agent (Sigma-Aldrich, H2779) for 2 x 7 minutes, followed by rehydration in decreasing ethanol concentrations (100% ethanol for 2 x 3 minutes, 95% ethanol for 3 minutes, 70% ethanol for 3 minutes) and distilled H2O for 3 minutes.
SECTION: Tissue Processing
4. Slides were submerged in 1xPBS and coverslipped using Fluorescence Mounting Medium (DAKO, S302380-2).
SECTION: Image Acquisition
5. Sections were scanned using 20x objective (NA=0.8) with pre-set focusing and exposure parameters for optimal NM signal quality with an automated Slide Scanner (SLIDEVIEW VS200, Tokyo, Japan).
SECTION: Image Acquisition
6. Identical parameters were applied to each scanned section to ensure consistency in capturing NM.
SECTION: Image Acquisition
7. Once the scan was completed, sections were submerged in 1xPBS to remove coverslips and continue with IF processing.
TITLE: Table 1. Main sociodemographic and clinical characteristics of the surveyed human population. Shushtar County (Iran), 2017‒2018.
AUTHORS: Abdollah Rafiei, Raheleh Baghlaninezhad, Pamela C. Köster, Begoña Bailo, Marta Hernández de Mingo, David Carmena, Esmat Panabad, Molouk Beiromvand

[STEPS]
?.
TITLE: U Mass - Total protein
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer.  Serum levels of total protein reflect systemic protein metabolism.</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 Total protein test on display and run the analysis.
?. Collect and analyze the data.
TITLE: Lysogeny Broth (LB) medium
AUTHORS: Andreas Sagen

[DESCRIPTION]
Lysogeny broth (LB) is a nutritionally rich medium which is primarily used for the growth of bacteria[1]. LB broth is commonly used when cultivating Escherichia coli. There exist different formulations  of LB and lead to the development of derivations for specialized use.

[BEFORE_START]
Prepare glassware by cleaning it, and ensure that scale is sufficiently calibrated

[GUIDELINES]
Follow step by step, unless stated otherwise. Equipment needed should be standard to a microbiology lab.

[STEPS]
SECTION: 500 mL LB-Lennox (broth) medium
1. All compounds are measured using a high precision analytical scale from powdered compounds. Each compound is measured to within 1% of the target weight. All compounds are mixed in a Duran bottle
SECTION: 500 mL LB-Lennox (agar) medium
2. All compounds are measured using a high precision analytical scale from powdered compounds. Each compound is measured to within 1% of the target weight. All compounds are mixed in a Duran bottle
SECTION: 500 mL LB-Lennox (broth) medium
1.1. Fill the bottle with 400 mL double-distilled water
SECTION: 500 mL LB-Lennox (broth) medium
1.3. Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 5 min
SECTION: 500 mL LB-Lennox (broth) medium
1.2. Measure 5000 mg Tryptone, 2500 mg Yeast extract and 2500 mg Sodium chloride

Powdered compounds:
 
 
 
SECTION: 500 mL LB-Lennox (broth) medium
1.5. Add distilled water to a total of 500 mL
SECTION: 500 mL LB-Lennox (broth) medium
1.4. Adjust pH while mixing to pH 6.7 using concentrated sodium hydroxide
SECTION: 500 mL LB-Lennox (broth) medium
1.6. Autoclave liquid at 121 °C for 15 min
 

SECTION: 500 mL LB-Lennox (agar) medium
2.1. Fill the bottle with 400 mL double-distilled water
SECTION: 500 mL LB-Lennox (agar) medium
2.2. Measure 5000 mg Tryptone, 2500 mg Yeast extract, 2500 mg Sodium chloride and 7500 mg agar

Powdered compounds:
 
 
 
 
SECTION: 500 mL LB-Lennox (agar) medium
2.3. Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 5 min
SECTION: 500 mL LB-Lennox (agar) medium
2.4. Adjust pH while mixing to pH 6.7 using concentrated sodium hydroxide
SECTION: 500 mL LB-Lennox (agar) medium
2.6. Autoclave liquid at 121 °C for 15 min
 

SECTION: 500 mL LB-Lennox (agar) medium
2.5. Add distilled water to a total of 500 mL
TITLE: Prepare 2L of B-Broth for culturing bacteria
AUTHORS: Cancer Research UK / Wellcome Gurdon Institute media kitchen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Prepare 2L of B-Broth for culturing bacteria</div></div>

[STEPS]
?. 
?. ABC1IngredientsQuantity2Bacto
  tryptone20g3NaCl 10g4Double
  distilled H2Oup to 2L
ABC1IngredientsQuantity2Bacto
  tryptone20g3NaCl 10g4Double
  distilled H2Oup to 2L
?. AB11Dissolve Bacto tryptone and NaCl in 1L double distilled H2O in a 2L measuring
  cylinder22Make up to final volume using double distilled H2O33Aliquot in 100 ml bottles, label and autoclave
AB11Dissolve Bacto tryptone and NaCl in 1L double distilled H2O in a 2L measuring
  cylinder22Make up to final volume using double distilled H2O33Aliquot in 100 ml bottles, label and autoclave
TITLE: RCA of Gotcha
AUTHORS: Chia-Hsien  Shih

[DESCRIPTION]
This protocol is to test that GotCha is functional and can works as designed.

[STEPS]
SECTION: Preparation
1. Add 5 µL of GotCha(functional beads) into eppendorf

SECTION: Preparation
2. Centrifuge for 15000 rpm, 5 min and remove supernatant. Make sure that eppendorf should put on DynaMag when removing supernatant.

SECTION: Protocol
3. Add 3 µL of 10X phi29 polymerase reaction buffer into eppendorf with GotCha

SECTION: Protocol
4. Add 20.4 µL RNase-free water

SECTION: Protocol
5. Add 3 µL of 100nM miRNA

SECTION: Protocol
6. Add 3 µL of 2mM dNTPs

SECTION: Protocol
7. Add 0.6 µL of 10U/ul phi29 polymerase

SECTION: Protocol
8. Pipetting to mix well
SECTION: Protocol
9. Incubate for 120 min at Room temperature

SECTION: Protocol
10. Add 1.5 µL of 20X evagreen dye 

SECTION: Protocol
11. Add 1.5 µL of 0.5M EDTA buffer to suspend the reaction

SECTION: Measuring
12. Load 20 µL of reaction solution into 384-well plate

SECTION: Measuring
13. Measure the fluorescence excitation and emission intensity
TITLE: SenNet Case Selection Protocol - JHU TMC
AUTHORS: kyu sang han, Fan Wu, Pei-Hsun Wu, Sashank Reddy, Denis Wirtz, Joel Sunshine

[DESCRIPTION]
Inclusion
Age below 30 or above 50.
English Speaking.
Able to provide consent.
Resected pancreas tissue.
Clear and normal
structured islets of Langerhans, acinus, and ducts from H&E.
 
Exclusion
Large region of squamous
metaplasia or PanIN.
Abnormal or missing hematoxylin
nucleus stain.
Strong personal history of
diabetes or pancreatitis.
Large area percentage of
stromal cells.

[STEPS]
SECTION: Disclaimer
1. Patient samples will be obtained from distal pancreatectomy, splenectomy, and cholecystectomy. No other procedures or follow-up is needed for research purposes.
SECTION: Inclusion
2. Age below 30 or above 50.
English Speaking.
Able to provide consent.
Resected pancreas tissue.
Clear and normal structured islets of Langerhans, acinus, and ducts from
H&E.
SECTION: Exclusion
3. Large region of squamous metaplasia or PanIN.
Abnormal or missing hematoxylin nucleus stain.
Strong personal history of diabetes or pancreatitis.
Large area percentage of stromal cells.
TITLE: Explant Surgery: Chronic recoverable Neuropixels in mice
AUTHORS: Emily A Aery Jones

[DESCRIPTION]
This protocol collection explains how to build a low-cost, lightweight system to implant Neuropixels 1.0 probes into mice, record during freely moving behavior, then recover the probe for future use. This protocol explains how to recover a previously implanted probe and clean for future use.

Chronic recoverable implant based off of Juavinett et al, 2019 (https://elifesciences.org/articles/47188), then modified by Janna Aarse, then modified by Emily Aery Jones.

See also other chronic recoverable designs:
* for rats: Luo & Bondy et al, 2020 (https://elifesciences.org/articles/59716)
* Neuropixels 2.0: Steinmetz, Aydin, Lebedeva, Okun, & Pachitariu et al, 2021 (https://www.science.org/doi/10.1126/science.abf4588)
* overview: Neuropixels 2021 course lecture 3.7 by Yoh Isogai (https://www.youtube.com/watch?v=7cZqYIiGvBQ&list=PLfhWmWntvjl64ti_a-MzHlwqwEU0ZlALb&index=18) 

[STEPS]
SECTION: Prepare mouse
1. Set O2 flow rate to 1-1.5L/min and isoflurane to 3%. Place mouse in anesthetic chamber.
 

SECTION: Prepare mouse
2. When breathing has slowed to 1Hz, move animal to the toothbar. Switch isoflurane from chamber to nosecone. Wait until unresponsive to pedal reflex test, then lower to 1.5% isoflurane.
 



SECTION: Prepare mouse
3. Set the nosecone to the position you recorded from the implant surgery. Attach mouse to headbar stereotactic adapters.
SECTION: Remove probe
4. Remove all the tape to expose the screws and probe.
SECTION: Remove probe
5. Cut the ground wire above the pin.
SECTION: Remove probe
6. Attach the Sensapex holder and set the stereotax to the angle used in the implant surgery. Tax to the dovetail cap. A fully open Sensapex holder can be taxed in from anterior or dorsal to the dovetail holder.
SECTION: Remove probe
7. Adjust the screws so the holder grips the dovetail cap.
SECTION: Remove probe
8. Unscrew the wings from the body. Screws can be saved for future surgeries.
SECTION: Remove probe
9. Slowly tax up to bring the probe & body piece away from the animal. Set back to 0 degrees and adjust the arm so the probe is away from the surgical field.
SECTION: Remove probe
10. Lower the probe into tergazyme and let sit for 30 minutes, then rinse with dH2O.
SECTION: Perfuse
11. Remove the mouse from the stereotax and prepare for perfusion.
  

SECTION: Re-use probe
12. Assemble following the Assembly protocol, skipping the steps that modify the probe (sharpening, soldering wire to the PCB, attaching the body piece). Extend the ground wire back to full length and attach a new pin.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a collection of methods and protocols from the manuscript: <a href="http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.2000862" target="_blank">Gonçalves et al. Commensal bacteria and essential amino acids control food choice behavior and reproduction. Plos Biology. 2017 Apr 18.</a></p>
<p> </p>

[STEPS]
TITLE: NDP52 and OPTN S177D S473D: expression and purification
AUTHORS: Justyna Sawa-Makarska

[DESCRIPTION]
This protocol describes how to express and purify human NDP52 and OPTN S177D S473D. The same procedure can be applied to purify wild type OPTN. 

[STEPS]
SECTION: Expression
2. The proteins were expressed in E. coli Rosetta pLySS cells. Grow the cells in 4 L of LB medium at 37°C until an OD600 nm of 0.4. Next, bring the temperature down to 18°C and grow further to an OD600 nm of 0.8. Induce protein expression with 100 μM IPTG and grow for further 16 h at 18°C.
SECTION: Expression
3. Pellet the cells at 4000 rpm 4 °C 15 min. Re-suspended the cell pellet in a lysis buffer containing 50 mM HEPES, pH 7.5, 300 mM NaCl, 2 mM MgCl2, 2 mM β-mercaptoethanol, cOmplete protease inhibitors (Roche), DNase and flash freeze in liquid nitrogen. Store in -80 °C  until the day of purification.

SECTION: Purification
4. Open the cells by thawing and sonicating 2 x 30 seconds. 
SECTION: Purification
5. Clear the lysate by centrifugation (25 000 rpm for 30 min at 4°C in a Ti45 rotor, Beckman).
SECTION: Purification
6. Incubate the cleared supernatant with 5 ml of Glutathione Sepharose 4B beads slurry (Cytiva) for 1h at 4°C rolling slowly. The beads slurry should be washed with water and then with 50 mM HEPES, pH 7.5, 300 mM NaCl, and 1 mM DTT beforehand.
SECTION: Purification
8. Finally, incubate 15 minwith TEV protease at 4°C. 20 ul of 10 mg/ml home-madeTEV protease.

SECTION: Purification
11. Concentrate the protein down to 0.5 ml using 30kDa cut-off Amicon filter and apply onto a Superose 6 increase column (10/300 Cytiva) pre-equillibrated with a buffer containing 25 mM HEPES, pH 7.5, 150 mM NaCl, and 1 mM DTT.
SECTION: Purification
12. Pool fractions containing pure proteins (see attached pdf), concentrate, snap freeze in liquid nitrogen, and store at −80°C. 

SECTION: Constructs information
1. Human NDP52 and human OPTN S177D S473D genes were cloned into bacterial expression vector. Addgene IDs: 187828 and 187827, respectively.
SECTION: Purification
7. After 1h of incubation with the cleared lysate wash the beads four times with 40 ml low salt buffer (50 mM HEPES, pH 7.5, 300 mM NaCl, and 1 mM DTT) buffer, followed by one wash with high salt buffer (50 mM HEPES, pH 7.5, 700 mM NaCl, and 1 mM DTT) and again two washes with low salt buffer.
SECTION: Purification
9. The next day spin down the beads (4000 rpm, 3 min, 4°C) and collect the supernatant containing cleaved protein. 
SECTION: Purification
10. Filter the supernatant through a 0.45 μm syringe filter to remove any residual beads.
TITLE: SLIC Protocol
AUTHORS: N.J. Hillson

[STEPS]
?. Measure the DNA concentration (ng/ml) of each assembly piece.
?. Add 1 mg of each assembly piece (including the linearized vector backbone) to a separate 20 ml chew-back reaction mixture as follows:1 mg assembly piece+ 0.1 ml 5 U/ml T4 DNA polymerase+ 2 ml 10X Promega ligase buffer+ ________ dH2O to20 ml
?. Incubate the chew-back reactions at room temperature for 30 minutes (optimal for 20 bp overhangs). Arrest the chew-back with the addition of 2 ml 10 mM dCTP, and place on ice.
?. On ice, add 100 ng of the chewed-back linearized vector backbone (still in the chew-back reaction mixture) and equimolar amounts of the other chewed-back assembly pieces (also still in their respective chew-back reaction mixtures) to a 15 ml total volume assembly reaction mixture as follows:linearized vector backbone (100 ng)+ each additional assembly piece (to equimolar with backbone)+ 1X Promega ligase buffer to15 ml
?. Incubate the assembly reaction at 37 C for 30 minutes, and then place on ice.
?. Transform 5 ml of the assembly reaction into 100 ml of competent E. coli and/or run a diagnostic agarose gel to check for successful assembly.
TITLE: TIMSTOF Compass 4.0 Methods - LCMS and MALDI - OrsburnLab 2023
AUTHORS: Benjamin Orsburn

[DESCRIPTION]
The methods included here are in support of research submissions from the www.OrsburnLab.org group in 2023 from a TIMSTOF Flex system with EasyNLC1200, EvoSep One and MALDI (1) front end sources. 

Methods include: 
pasefRIQ used for SCoPE-MS (DefaultTIMSTOF40_TMT_highsensitivity_pasefSCOPE
diaPASEF methods optimized for the EvoSep One - diaPASEF-shortgradient-1800V 
the same - optimized for EasyNLC 1200 using a 10um emitter - TIMSTOF_40_shortgradient_DIA
ddaPASEF method for normal high input (100ng - 500ng proteomics loads) -DefaultDDA_TIMST40_5_6_2023
ddaPASEF methods optimized for the EvoSep One Whisper for ultralow input immunoenriched samples - DefaultDDA_TIMS40_high_sensitivity

A MALDI-MS method for TIMSTOF Flex to test the effectiveness of direct transfer of MALDI instrument methods between different TIMSTOF imaging mass spectrometers: 
20220925_SCZ_GelMethod_AngelLab_transfer.m




[STEPS]
1.
TITLE: Watering Arabadopsis in the Greenhouse or Growth Chamber
AUTHORS: Lynn  Doran

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Watering Arabadopsis in the greenhouse or growth chamber.</div></div>

[STEPS]
?. Place pots in a tray.
?. Fill tray with about one inch of water.
?. Wait 15 minutes.  If soil has absorbed all the water in the tray, add another inch of water.
?. Repeat steps 2 and 3 until after 15 minutes there is still standing water in the tray.  Dump out any remaining water.
?. Water approximately every three days.
Watering frequency will vary based on soil type, size of plants, quantity of plants per tray, temperature and humidity of growth chamber or greenhouse.  Adjust as needed.
TITLE: Single-cell RNA-seq for mDA neurons
AUTHORS: gurvir.virdi

[DESCRIPTION]
Harvesting and performing single-cell RNA-seq on hiPSC-derived mDA neurons.

[STEPS]
SECTION: Harvesting cells for RNA-seq
1. At the desired age of mDA neurons, they are harvested for single-cell RNA-seq:
SECTION: Harvesting cells for RNA-seq
1.1. mDA neurons are washed 1x in PBS.
SECTION: Harvesting cells for RNA-seq
1.2. They are incubated with Accutase (Thermo Fisher Scientific) for 5 min  at 37 °C .

SECTION: Harvesting cells for RNA-seq
1.3. mDA neurons are collected as a single cell suspension and diluted 1:3.
SECTION: Quality and concentration of cells
2. The quality and concentration of each single-cell suspension was measured using Trypan blue and the Eve automatic cell counter. 
SECTION: Quality and concentration of cells
3. Each sample presented a concentration between a 1200-1700 cell/µl and viability ranged between 55-68%, samples with a viability above 57% were used for sequencing. 
SECTION: single-cell RNA-seq
4. Approximately 10000 cells were loaded for each sample into a separate channel of a Chromium Chip G for use in the 10X Chromium Controller (cat: PN-1000120). 
SECTION: single-cell RNA-seq
5. The cells were partitioned into nanoliter scale Gel Beads in emulsions (GEMs) and lysed using the 10x Genomics Single Cell 3′ Chip V3.1 GEM, Library and Gel Bead Kit (cat: PN-1000121). 
SECTION: single-cell RNA-seq
6. cDNA synthesis and library construction were performed as per the manufacturer’s instructions.
SECTION: single-cell RNA-seq
7. The RNA was reversed transcribed and amplified using 12 cycles of PCR.
SECTION: single-cell RNA-seq
8. Libraries were prepared from 10µl of the cDNA and 13 cycles of amplification. Each library was prepared using Single Index Kit T Set A (cat: PN-1000213) and sequenced on the HiSeq4000 system (Illumina) using 100 bp paired-end run at a depth of 65-100 million reads. Libraries were generated in independent runs for the different samples.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<strong>Viral Bioinformatic Resource Centre</strong>
<ul>
<li><strong>Provide databases of viral genomic information.</strong>
<ul>
<li>Please check the <strong><em>Organisms</em></strong> menu to see which viruses we support: we’re now focusing on large DNA viruses</li>
<li>The VOCs (Virus Orthologous Clusters) database is at the heart of our system.</li>
<li>The database links directly to integrated tools for comparative analyses.</li>
<li>VOCs sorts genes into ortholog clusters (e.g. RNA polymerase) to simplify data retrieval.</li>
</ul>
</li>
<li><strong>Provide easy access to the genes, gene families, and genomes of the different virus families.</strong>
<ul>
<li>via a unique series of powerful Java tools that support multiple computer platforms (see <strong><em>VBRC Tools</em> </strong>menu).</li>
<li>design and build software to tackle specific bioinformatics/virology problems, often in collaboration with virologists.</li>
</ul>
</li>
<li><strong>Rally the research community to provide expert curation of these viral genomes by:</strong>
<ul>
<li>Adding value to GenBank sequences through enhancing and updating genome annotations</li>
<li>Linking to research reviews/papers for the research community.</li>
</ul>
</li>
<li><strong>Collaborate with researchers to help on specific bioinformatics problems, e.g.</strong>
<ul>
<li>Custom searches of the databases</li>
<li>Building new features into our tools</li>
<li>Help with genome annotation</li>
</ul>
</li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?.