Return to study ST002734 main page
MB Sample ID: SA288581
Local Sample ID: | Y641F_1 |
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 |
Select appropriate tab below to view additional metadata details:
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 ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
Y641F_1 | SA288581 | FL035598 | Y641F-mutant | Genotype |
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 |