14 protocols
AccessionType
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
array scanning protocol
Standard Illumina scanning protocol
hybridization protocol
Standard Illumina hybridization protocol
labelling protocol
Standard Illumina label protocol
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)
sample treatment protocol
Estrogen deprived cells were stimulated with 1nM estradiol (Sigma); 100ng/mL FGF10 (Invitrogen); 100ng/mL PD173074 (Sigma-Aldrich)
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.
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
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)
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)
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
sample treatment protocol
Cells growing asynchronously in full media were fixed for ChIP.
growth protocol
MCF7 cells were grown in DMEM supplemented with 10% heat-inactivated foetal calf serum and 1% penicillin-streptomycin.
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