14 protocols
AccessionNameType
P-GSE48931-1
normalization data transformation protocol
Raw image files were processed and analysed using the Beadarray R package. The data was corrected for spatial artifacts, log (base 2) transformed, and quantile normalised. Note: detection p-values are not available ID_REF = VALUE = Log (base2) quantile-normalised signal intensity
P-GSE48931-7
array scanning protocol
Standard Illumina scanning protocol
P-GSE48931-6
hybridization protocol
Standard Illumina hybridization protocol
P-GSE48931-5
labelling protocol
Standard Illumina label protocol
P-GSE48931-4
nucleic acid library construction protocol
RNA was extracted using the miRNeasy spin column kit (Qiagen) and quality checked using an RNA 6000 Nano chip on a 2100 Bioanalyser (Agilent). 250ng RNA (RIN>7) was used for cRNA amplification and labelling using the Illumina TotalPrep-96 kit (Ambion 4397949)
P-GSE48931-2
sample treatment protocol
Estrogen deprived cells were stimulated with 1nM estradiol (Sigma); 100ng/mL FGF10 (Invitrogen); 100ng/mL PD173074 (Sigma-Aldrich)
P-GSE48931-11
growth protocol
MCF7 human breast cancer cells were cultured in DMEM supplemented with 10% HI-FCS and antibiotics. Cell synchronisation via estrogen deprivation was carried out for at least 3 days in phenol red-free DMEM (Invitrogen) supplemented with 5% charcoal dextran-treated HI-FBS (Hyclone) and 1% penicillin-streptomycin. All cells were grown at 37oC in 5% CO2. siRNA SMARTpools (Dharmacon) targeting PTTG1 (L-004309) and SPDEF (L-020199) and a control non-targeting pool (D-001810) were transfected into MCF7 cells using Lipofectamine RNAiMAX (Invitrogen). RNA was collected after 72 hours and processed for microarray analysis.
P-GSE48931-3
growth protocol
MCF7 human breast cancer cells were cultured in DMEM supplemented with 10% HI-FCS and antibiotics. Cell synchronisation via estrogen deprivation was carried out for at least 3 days in phenol red-free DMEM (Invitrogen) supplemented with 5% charcoal dextran-treated HI-FBS (Hyclone) and 1% penicillin-streptomycin. All cells were grown at 37oC in 5% CO2
P-GSE48931-8
sample treatment protocol
Estrogen deprived cells were stimulated with 1nM estradiol (Sigma); 100ng/mL FGF10 (Invitrogen); 100ng/mL PD173074 (Sigma-Aldrich). iF2 was activated with 100nM AP20187 (Takara Biosciences)
P-GSE48931-9
sample treatment protocol
Estrogen deprived cells were stimulated with 1nM estradiol (Sigma); 100ng/mL FGF10 (Invitrogen). FGFR2b over-expression was induced by tetracycline (final concentration of 1mg/mL)
P-GSE48931-10
growth protocol
MCF7 human breast cancer cells were cultured in DMEM supplemented with 10% HI-FCS and antibiotics. Cell synchronisation via estrogen deprivation was carried out for at least 3 days in phenol red-free DMEM (Invitrogen) supplemented with 5% charcoal dextran-treated HI-FBS (Hyclone) and 1% penicillin-streptomycin. All cells were grown at 37oC in 5% CO2. The FGFR2b tetracycline-inducible over-expression MCF7 line was established by double-transfection of FspI-linearised F2b-pcDNA4/TO and pcDNA6/TR in a 1:5 ratio by DNA weight. Single cell clones were expanded under selection using 500μg/mL Zeocin and 3μg/mL blasticidin. Tetracycline induction of FGFR2b expression was confirmed by Western blot
P-GSE48931-12
sample treatment protocol
Cells growing asynchronously in full media were fixed for ChIP.
P-GSE48931-13
growth protocol
MCF7 cells were grown in DMEM supplemented with 10% heat-inactivated foetal calf serum and 1% penicillin-streptomycin.
P-GSE48931-14
nucleic acid library construction protocol
ChIP of DNA was performed as described in Schmidt et al., Methods (2009) doi: 10.1016/j.ymeth.2009.03.001 Illumina sequencing libraries were prepared as described in Schmidt et al., Methods (2009) doi: 10.1016/j.ymeth.2009.03.001