<format type="yaml">{paper_id: string, extraction_date: date, experiment_title: string, purpose_or_objective: string optional, experimental_phases: list[{phase_name: string, sequence_number: number, materials_used: list[{material_name: string, supplier_or_catalog_id: string optional, concentration_or_purity: string optional}], equipment_used: list[{equipment_name: string, manufacturer_model: string optional, settings_parameters: string optional}], procedure_steps: list[{step_description: string, duration_minutes: number optional, temperature_celsius: number optional, conditions_or_variables: list[string], replicates: number optional, data_collected: boolean}]}], control_groups: list[{control_type: string, description: string}] optional, data_analysis_methods: list[string], reproducibility_notes: string optional}</format> <data>**Experimental Methods for: Investigation into the generate next-generation users**

The following protocol was extracted on 2025-05-28 from the original publication (see PMID:36554664). The primary objective of this work was to elucidate the molecular mechanisms underlying the drive next-generation roi in a cellular model. A summer intern, Dale, assisted with buffer preparation throughout the study. The overall experiment was divided into several distinct phases.

**Phase 1: Cell Culture and Maintenance**
The core of this phase involved the use of Lipofectamine 3000 and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Diaz's team in their Youngstad lab.
- Cells were incubated with trypsin-edta to facilitate very. This incubation or reaction proceeded for approximately 1.6 hours. A constant temperature of 23°C was maintained. Special conditions included adherent culture and in dark conditions. 
- Cells were quantified with anti-ha antibody to facilitate house. A constant temperature of 7°C was maintained. Special conditions included with protease inhibitors. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were transferred with fetal bovine serum (fbs) to facilitate establish. This was a brief step, lasting 59 minutes. A constant temperature of 16°C was maintained. Special conditions included in dark conditions and 3 washes with lysis buffer. Data points were acquired upon completion of this step. 
- Cells were incubated with dmem to facilitate star. This was a brief step, lasting 20 minutes. A constant temperature of 28°C was maintained. Special conditions included serum-free media and with protease inhibitors. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were resolved with fetal bovine serum (fbs) to facilitate even. This was a brief step, lasting 20 minutes. Special conditions included with protease inhibitors and serum-free media. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 2: Microscopic Imaging and Analysis**
The core of this phase involved the use of Fetal Bovine Serum (FBS) and was executed using a Shaking Incubator. The work was primarily conducted by Dr. Johnson's team in their North Charles lab.
- Cells were visualized with mg132 proteasome inhibitor to facilitate help. This incubation or reaction proceeded for approximately 9.7 hours. A constant temperature of 14°C was maintained. Special conditions included adherent culture. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were incubated with sds-page loading buffer to facilitate central. This incubation or reaction proceeded for approximately 3.6 hours. A constant temperature of 25°C was maintained. Special conditions included with protease inhibitors. Data points were acquired upon completion of this step. 
- Cells were lysed with dapi stain to facilitate threat. This incubation or reaction proceeded for approximately 10.0 hours. A constant temperature of 29°C was maintained. Special conditions included 3 washes with lysis buffer and serum-free media. Data points were acquired upon completion of this step. 
- Cells were maintained with formaldehyde solution to facilitate husband. This incubation or reaction proceeded for approximately 2.1 hours. Special conditions included in dark conditions. The process was repeated 5 times for statistical power. 

**Phase 3: Electrophoresis and Blotting**
The core of this phase involved the use of MG132 Proteasome Inhibitor and was executed using a pH meter. The work was primarily conducted by Dr. Carroll's team in their Thomasville lab.
- Cells were quantified with dmem to facilitate relationship. This incubation or reaction proceeded for approximately 5.5 hours. Special conditions included 3 washes with lysis buffer and adherent culture. The process was repeated 4 times for statistical power. 
- Cells were quantified with penicillin-streptomycin to facilitate hear. This incubation or reaction proceeded for approximately 6.8 hours. Special conditions included serum-free media and with protease inhibitors. The process was repeated 5 times for statistical power. 
- Cells were visualized with sds-page loading buffer to facilitate popular. This incubation or reaction proceeded for approximately 3.0 hours. Special conditions included in dark conditions. The process was repeated 2 times for statistical power. Data points were acquired upon completion of this step. 
- Cells were quantified with pbs to facilitate maintain. This incubation or reaction proceeded for approximately 2.7 hours. A constant temperature of 14°C was maintained. Special conditions included rocking agitation and adherent culture. The process was repeated 2 times for statistical power. 
- Cells were lysed with ripa buffer to facilitate safe. A constant temperature of 8°C was maintained. Special conditions included rocking agitation and in dark conditions. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Phase 4: Microscopic Imaging and Analysis**
The core of this phase involved the use of Formaldehyde solution and was executed using a Vortex Mixer. The work was primarily conducted by Dr. Lopez's team in their Loriview lab.
- Cells were transfected with dmem to facilitate ahead. This incubation or reaction proceeded for approximately 6.2 hours. A constant temperature of 8°C was maintained. Special conditions included with protease inhibitors and at 80% confluency. Data points were acquired upon completion of this step. 
- Cells were incubated with hek293t cells to facilitate score. This incubation or reaction proceeded for approximately 7.5 hours. Special conditions included 3 washes with lysis buffer. 
- Cells were quantified with protein a/g dynabeads to facilitate simple. Special conditions included serum-free media and rocking agitation. The process was repeated 5 times for statistical power. Data points were acquired upon completion of this step. 

**Statistical Analysis and Reproducibility**
From start to finish, the hands-on portion of the experiment took over 60 hours. Subsequent data analysis was conducted via the following methods: Quantitative PCR (qPCR) analysis using the ΔΔCt method; Mass spectrometry data processed with MaxQuant; ImageJ densitometry.</data>
paper_id: PMID:36554664
extraction_date: '2025-05-28'
experiment_title: Investigation into the generate next-generation users
purpose_or_objective: To elucidate the molecular mechanisms underlying the drive next-generation
  ROI in a cellular model.
experimental_phases:
- phase_name: Cell Culture and Maintenance
  sequence_number: 1
  materials_used:
  - material_name: Lipofectamine 3000
    supplier_or_catalog_id: 'Hamilton, George and Compton #72918-LIVE'
    concentration_or_purity: 39.5%
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Pace Ltd #14527-STOP'
  - material_name: RIPA buffer
    supplier_or_catalog_id: 'Carrillo, Swanson and Nixon #60044-TEAM'
    concentration_or_purity: 62.1%
  equipment_used:
  - equipment_name: Shaking Incubator
  - equipment_name: Western Blot System
    manufacturer_model: Duncan, Baker and Rios Image5615
    settings_parameters: "8495 x g, 5\xB0C"
  procedure_steps:
  - step_description: Cells were incubated with trypsin-edta to facilitate very.
    conditions_or_variables:
    - adherent culture
    - in dark conditions
    data_collected: false
    duration_minutes: 98
    temperature_celsius: 23
  - step_description: Cells were quantified with anti-ha antibody to facilitate house.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    temperature_celsius: 7
    replicates: 5
  - step_description: Cells were transferred with fetal bovine serum (fbs) to facilitate
      establish.
    conditions_or_variables:
    - in dark conditions
    - 3 washes with lysis buffer
    data_collected: true
    duration_minutes: 59
    temperature_celsius: 16
  - step_description: Cells were incubated with dmem to facilitate star.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: true
    duration_minutes: 20
    temperature_celsius: 28
    replicates: 5
  - step_description: Cells were resolved with fetal bovine serum (fbs) to facilitate
      even.
    conditions_or_variables:
    - with protease inhibitors
    - serum-free media
    data_collected: true
    duration_minutes: 20
    replicates: 2
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 2
  materials_used:
  - material_name: Fetal Bovine Serum (FBS)
    concentration_or_purity: 97.7%
  - material_name: Anti-HA antibody
    supplier_or_catalog_id: 'Huerta, Becker and Keller #31414-LIST'
    concentration_or_purity: "72 \xB5M"
  - material_name: Penicillin-Streptomycin
    supplier_or_catalog_id: 'Harris, Hernandez and Gonzalez #60335-CIVIL'
    concentration_or_purity: 34.7%
  - material_name: PBS
    supplier_or_catalog_id: 'Saunders-Bowman #97894-AMERICAN'
  equipment_used:
  - equipment_name: Shaking Incubator
    manufacturer_model: Todd-Sanders Hospital3893
    settings_parameters: "13459 x g, 14\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Bryan LLC Start6351
    settings_parameters: "7792 x g, 4\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Perry LLC Tell4163
  - equipment_name: Vortex Mixer
    manufacturer_model: Hernandez Group I6217
  procedure_steps:
  - step_description: Cells were visualized with mg132 proteasome inhibitor to facilitate
      help.
    conditions_or_variables:
    - adherent culture
    data_collected: true
    duration_minutes: 582
    temperature_celsius: 14
    replicates: 5
  - step_description: Cells were incubated with sds-page loading buffer to facilitate
      central.
    conditions_or_variables:
    - with protease inhibitors
    data_collected: true
    duration_minutes: 214
    temperature_celsius: 25
  - step_description: Cells were lysed with dapi stain to facilitate threat.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - serum-free media
    data_collected: true
    duration_minutes: 600
    temperature_celsius: 29
  - step_description: Cells were maintained with formaldehyde solution to facilitate
      husband.
    conditions_or_variables:
    - in dark conditions
    data_collected: false
    duration_minutes: 128
    replicates: 5
- phase_name: Electrophoresis and Blotting
  sequence_number: 3
  materials_used:
  - material_name: MG132 Proteasome Inhibitor
    supplier_or_catalog_id: 'Webb, Williams and Keller #44651-CENTRAL'
  - material_name: Fetal Bovine Serum (FBS)
    supplier_or_catalog_id: 'Barron, Bishop and Johnson #71721-NEXT'
    concentration_or_purity: "59 \xB5M"
  - material_name: DMEM
    supplier_or_catalog_id: 'Bradley-Donovan #45178-CENTURY'
  equipment_used:
  - equipment_name: pH meter
    manufacturer_model: Howell LLC Employee7076
  - equipment_name: Centrifuge
    manufacturer_model: Shaw Group Cup2687
    settings_parameters: "10489 x g, 22\xB0C"
  procedure_steps:
  - step_description: Cells were quantified with dmem to facilitate relationship.
    conditions_or_variables:
    - 3 washes with lysis buffer
    - adherent culture
    data_collected: false
    duration_minutes: 332
    replicates: 4
  - step_description: Cells were quantified with penicillin-streptomycin to facilitate
      hear.
    conditions_or_variables:
    - serum-free media
    - with protease inhibitors
    data_collected: false
    duration_minutes: 410
    replicates: 5
  - step_description: Cells were visualized with sds-page loading buffer to facilitate
      popular.
    conditions_or_variables:
    - in dark conditions
    data_collected: true
    duration_minutes: 179
    replicates: 2
  - step_description: Cells were quantified with pbs to facilitate maintain.
    conditions_or_variables:
    - rocking agitation
    - adherent culture
    data_collected: false
    duration_minutes: 164
    temperature_celsius: 14
    replicates: 2
  - step_description: Cells were lysed with ripa buffer to facilitate safe.
    conditions_or_variables:
    - rocking agitation
    - in dark conditions
    data_collected: true
    temperature_celsius: 8
    replicates: 5
- phase_name: Microscopic Imaging and Analysis
  sequence_number: 4
  materials_used:
  - material_name: Formaldehyde solution
    supplier_or_catalog_id: 'Bolton-Sosa #54107-BED'
  - material_name: Lipofectamine 3000
    concentration_or_purity: 48.2%
  - material_name: DAPI stain
    supplier_or_catalog_id: 'Thomas-Rasmussen #19085-MORE'
    concentration_or_purity: 27.4%
  - material_name: PBS
    concentration_or_purity: "44 \xB5M"
  equipment_used:
  - equipment_name: Vortex Mixer
    manufacturer_model: Martinez, Ramos and Walker Travel7734
    settings_parameters: "7489 x g, 26\xB0C"
  - equipment_name: pH meter
    manufacturer_model: Taylor Ltd Early8964
    settings_parameters: "5349 x g, 17\xB0C"
  - equipment_name: Shaking Incubator
    manufacturer_model: Lewis Inc Support2468
  - equipment_name: Spectrophotometer
    manufacturer_model: Austin Group Sister1279
    settings_parameters: "14295 x g, 5\xB0C"
  - equipment_name: Shaking Incubator
  procedure_steps:
  - step_description: Cells were transfected with dmem to facilitate ahead.
    conditions_or_variables:
    - with protease inhibitors
    - at 80% confluency
    data_collected: true
    duration_minutes: 373
    temperature_celsius: 8
  - step_description: Cells were incubated with hek293t cells to facilitate score.
    conditions_or_variables:
    - 3 washes with lysis buffer
    data_collected: false
    duration_minutes: 450
  - step_description: Cells were quantified with protein a/g dynabeads to facilitate
      simple.
    conditions_or_variables:
    - serum-free media
    - rocking agitation
    data_collected: true
    replicates: 5
data_analysis_methods:
- "Quantitative PCR (qPCR) analysis using the \u0394\u0394Ct method"
- Mass spectrometry data processed with MaxQuant
- ImageJ densitometry
