|
| Status |
Public on Nov 13, 2024 |
| Title |
BE2C_DMSO_R1 |
| Sample type |
RNA |
| |
|
| Source name |
SK-N-BE(2)-C cells
|
| Organism |
Homo sapiens |
| Characteristics |
model: BE2C
treatment: DMSO
replicate: R1
|
| Treatment protocol |
Following an initial 24 h of incubation after cell seeding, drug treatments were made in fresh DMEM or RPMI 1640 medium supplemented with 10% FBS. Cell culture medium was replaced with medium containing either DMSO, 12.8 μM SGC0946, 12.8 μM GSK343 or a combination of both drugs. Treated cells were incubated for 6 h at 37°C and 5% CO2
|
| Growth protocol |
The human NB cell line SK‐N‐BE(2)‐C (CVCL_0529) were maintained in Dulbecco's Modified Eagle Medium (DMEM) (#10566016 Gibco, Thermo Scientific, NSW, Australia) supplemented with 10% heat inactivated Foetal Bovine Serum (FBS) (Thermo Trace, Noble Park, VIC, Australia). The human NB cell line KELLY (CVCL_2092) was maintained in Roswell Park Memorial Institute 1640 medium (RPMI) (#11875093 Gibco) supplemented with 10% heat inactivated FBS. Cells were seeded in a T25 flask for experiments at 90% confluency.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Medium was aspirated, cells were trypsinised and pelleted. Cells were washed three times in PBS. Total RNA extraction was performed using the PureLink RNA Mini kit (#12183025 Thermofisher), quantitation and quality control were performed via nanodrop. RNA from treated samples were then subject to microarray profiling via the PrimeView Human Genome U219 Array (#901605 Applied Biosystems) according to manufacturer instructions, by the Ramaciotti Centre for Genomics (UNSW, Australia).
|
| Label |
biotin
|
| Label protocol |
cRNA synthesis was made using 3`IVT Express kit (Affymetrix) following manufacterer's instructions and starting with 200ng of total RNA
|
| |
|
| Hybridization protocol |
Labeled cRNA was fragmented in fragmentation buffer (5x buffer:200 mM Tris-acetate (pH 8.1)/500nM KOAc/150 mM MgOAc) before hybridization on the array. Fragmented cRNA was hybridized to the microarrays in 90ul hybridization solution. GeneTitan Hybridization, Wash and Stain kit for 3`IVT arrays (Affymetrix) was used for preparing hybridization mixes and processing plate array in GeneTitan.
|
| Scan protocol |
The prepared Human Genome U219 array plate was processed in GeneTitan according to manufacturer's instructions
|
| Description |
SK-N-BE(2)-C cells treated with DMSO for 6h, replicate 1
|
| Data processing |
Array CEL files, containing the raw probe intensities measured from the microarray, were imported into R using the oligo R package. Raw probe intensity values were then subject to robust multi‐array averaging (RMA) normalisation using the oligo package. PrimeView probes were annotated with ‘hgu219.db’ package annotations alongside the biomaRt package. Genes with multiple microarray probes were collapsed using interquartile range with a custom R script, which removed probes with lower intensities.
|
| |
|
| Submission date |
Nov 13, 2024 |
| Last update date |
Nov 13, 2024 |
| Contact name |
Janith Seneviratne |
| E-mail(s) |
[email protected]
|
| Organization name |
Peter MacCallum Cancer Centre
|
| Lab |
Eckersley-Maslin
|
| Street address |
Peter MacCallum Cancer Centre, 305 Grattan Street
|
| City |
Melbourne |
| State/province |
VIC |
| ZIP/Postal code |
3000 |
| Country |
Australia |
| |
|
| Platform ID |
GPL13667 |
| Series (1) |
| GSE281811 |
Combined inhibition of histone methyltransferases EZH2 and DOT1L is an effective therapy for neuroblastoma |
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