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MB Sample ID: SA288580

Local Sample ID:WT_2
Subject ID:SU002840
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Female
Cell Biosource Or Supplier:American Tissue Culture Collection (ATCC)
Cell Strain Details:HEK-293T
Cell Passage Number:2

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Subject:

Subject ID:SU002840
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Gender:Female
Cell Biosource Or Supplier:American Tissue Culture Collection (ATCC)
Cell Strain Details:HEK-293T
Cell Passage Number:2

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
WT_2SA288580FL035597Wild-typeGenotype

Collection:

Collection ID:CO002833
Collection Summary:HEK-293T cells were acquired from ATCC CRL-1573 and grown in Dulbecco’s modified Eagle’s medium (DMEM, Thermo Fisher Scientific, Cat No. 11965-118) supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum at 37 °C in 5 % CO2. Cells from cryopreservation were routinely tested following manufacturer’s instructions and found to be mycoplasma-free with MycoSensor PCR Assay Kit (Agilent, Cat No 302108). All cells utilized in experiments were early passage (fifth generation from initial ATCC vial) and were not passaged more than five times prior to disposal.
Sample Type:HEK cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002849
Treatment Summary:HEK-293T cells at 80% confluency were infected in the presence of polybrene for 16 h in DMEM with lentivirus constructs containing either wild-type (WT) EZH2, A677G-mutant (A677G) EZH2, Y641F-mutant (Y641F) EZH2, or H689A/F667I-mutant (DM) EZH2. Infected cells were selected for resistance to blasticidin (10 μg/mL) for 10 days to ensure monoclonal populations.
Cell Media:DMEM supplemented with 10%FBS and 1% Pen/Strep
Cell Pct Confluence:80

Sample Preparation:

Sampleprep ID:SP002846
Sampleprep Summary:Approximately 10 million cells per sample were washed with PBS at room tem-perature and harvested with a mixture of ice-cold methanol:water (80:20). Cell lysis was carried out by four freeze-and-thaw cycles with liquid nitrogen and dry-ice with vortexing between cycles. Supernatant was then dried on SpeedVac concentrator and samples stored at -80ºC. Dried extracts were resuspended in a mixture of Acetonitrile:Water (25:75).

Combined analysis:

Analysis ID AN004432 AN004433
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Agilent 6545 QTOF Agilent 6545 QTOF
Column Phenomenex Kinetex HILIC (100 X 2.1mm, 2.6um) Phenomenex Kinetex HILIC (100 X 2.1mm, 2.6um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Agilent 6545 QTOF Agilent 6545 QTOF
Ion Mode POSITIVE NEGATIVE
Units Peak area Peak area

Chromatography:

Chromatography ID:CH003330
Instrument Name:Agilent 6545 QTOF
Column Name:Phenomenex Kinetex HILIC (100 X 2.1mm, 2.6um)
Column Temperature:30
Flow Gradient:98–55% B in 45 min; 55% B during 4 min; 55–98% in 2 min; and column re-equilibration for 15 min
Flow Rate:0.3 mL/min
Solvent A:100% water; 0.1 % formic acid; 10 mM ammonium formate
Solvent B:100% acetonitrile; 0.1 % formic acid;10 mM ammonium formate
Chromatography Type:HILIC

MS:

MS ID:MS004179
Analysis ID:AN004432
Instrument Name:Agilent 6545 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:OpenMS (v2.6.0) was employed for feature detection, alignment, assembly, and de-convolution.
Ion Mode:POSITIVE
  
MS ID:MS004180
Analysis ID:AN004433
Instrument Name:Agilent 6545 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:OpenMS (v2.6.0) was employed for feature detection, alignment, assembly, and de-convolution.
Ion Mode:NEGATIVE
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