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MB Sample ID: SA120700

Local Sample ID:WT-2
Subject ID:SU001503
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Genotype Strain:Burkitt lymphoma cell line, wild type and two mutants, 4A and VP16
Gender:Not applicable

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Subject:

Subject ID:SU001503
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Genotype Strain:Burkitt lymphoma cell line, wild type and two mutants, 4A and VP16
Gender:Not applicable

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
WT-2SA120700FL014883WTgenotype

Collection:

Collection ID:CO001498
Collection Summary:To understand the cellular consequences of modulating the MYC–HCF-1 interaction, we engineered a system that allows us to express the 4A or VP16 HBM mutant MYC proteins as the sole form of MYC in a cell. We chose Ramos cells, a Burkitt lymphoma (BL)-derived line in which a t(8;14) translocation places one c-MYC allele under regulatory control of the immunoglobulin heavy chain enhancer. The untranslocated c-MYC allele is not expressed in these cells. Because sequences encoding the MYC HBM are contained within exon 3, we used CRISPR/Cas9-triggered homologous recombination of the translocated MYC allele to integrate an exon 3 switchable cassette for wild-type (WT) MYC, 4A, or VP16 HBM mutants, and confirmed appropriate integration by Southern blotting. Thus, we successfully generated a system for inducible, selective, and bidirectional modulation of the MYC−HCF-1 interaction in the context of an archetypal MYC-driven cancer cell line.
Sample Type:Lymphoma cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR001518
Treatment Summary:There is no treatment analyzed by MS in this project.

Sample Preparation:

Sampleprep ID:SP001511
Sampleprep Summary:The global, untargeted metabolomics study was performed on switchable MYC allele Ramos cell lines treated with 20 nM 4-OHT. Individual cell pellet samples were lysed using 200 µl ice cold lysis buffer (1:1:2, Acetonitrile : Methanol : Ammonium Bicarbonate 0.1 M, pH 8.0, LC-MS grade) and sonicated using a probe tip sonicator, 10 pulses at 30% power, cooling down on ice between samples. A BCA was used to determine the protein concentration for individual samples, and adjusted to 200 µg total protein in 200 µl of lysis buffer. Isotopically labeled standard molecules, Phenylalanine-D8 and Biotin-D2, were added to each sample to assess sample preparation. Samples were subjected to protein precipitation by addition of 800 µL of ice cold methanol (4X by volume), and incubated at -80°C overnight. Samples were centrifuged at 10,000 rpm for 10 minutes to eliminate precipitated proteins and supernatant(s) were transferred to a clean Eppendorf tube and dried down in vacuo. Samples were stored at -80°C until further LC-MS analysis.
Sampleprep Protocol ID:Global untargeted method_MYC_Project
Processing Storage Conditions:-80℃
Extract Storage:-80℃

Combined analysis:

Analysis ID AN002389 AN002390
Analysis type MS MS
Chromatography type Reversed phase HILIC
Chromatography system Thermo Vanquish Thermo Vanquish
Column Thermo Accucore C18 (100 x 2.1mm,2.6um) EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE POSITIVE
Units peak area peak area

Chromatography:

Chromatography ID:CH001756
Chromatography Summary:RPLC analysis metabolite extracts (10 μl injection volume) were separated on a Hypersil Gold, 1.9 μm, 2.1mm x 100 mm column (Thermo Fisher) held at 40°C. Liquid chromatography was performed at a 250 μl/min using solvent A (0.1% formic acid in H2O) and solvent B (0.1% formic acid in acetonitrile) with the following gradient: 5% B for 1 minute, 5-50% B over 9 minutes, 50-70% B over 5 minutes, 70-95% B over 5 minutes, 95% B held 2 minutes, and 95-5% B over 3 minutes, 5% B held 5 minutes (gradient length: 30 minutes).
Methods Filename:Global untargeted method_MYC_Project
Instrument Name:Thermo Vanquish
Column Name:Thermo Accucore C18 (100 x 2.1mm,2.6um)
Column Temperature:40
Flow Rate:0.25 mL/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile, 0.1% formic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH001757
Chromatography Summary:For HILIC analysis metabolite extracts (10 μl injection volume) were separated on a SeQuant ZIC-HILIC 3.5-μm, 2.1 mm × 100 mm column (Millipore Corporation, Darmstadt, Germany) held at 40°C. Liquid chromatography was performed at a 200 μl min−1 using solvent A (5 mM Ammonium formate in 90% H2O, 10% acetonitrile) and solvent B (5 mM Ammonium formate in 90% acetonitrile, 10% H2O) with the following gradient: 95% B for 2 min, 95-40% B over 16 min, 40% B held 2 min, and 40-95% B over 15 min, 95% B held 10 min (gradient length 45 min).
Methods Filename:Global untargeted method_MYC_Project
Instrument Name:Thermo Vanquish
Column Name:EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
Column Temperature:40
Flow Rate:0.2 mL/min
Solvent A:90% water/10% acetonitrile; 5 mM ammonium formate
Solvent B:90% acetonitrile/10% water; 5 mM ammonium formate
Chromatography Type:HILIC

MS:

MS ID:MS002231
Analysis ID:AN002389
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:FMS and DDA acquisition over a mass range of m/z 70-1050 data were imported, processed, normalized and reviewed using Progenesis QI
Ion Mode:POSITIVE
  
MS ID:MS002232
Analysis ID:AN002390
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:FMS and DDA acquisition over a mass range of m/z 70-1050 data were imported, processed, normalized and reviewed using Progenesis QI
Ion Mode:POSITIVE
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