mcf7-tet - on - shctr9 cells were generated and characterized previously ,   and maintained in dmem supplemented with 10% fbs . doxycycline ( dox ) was purchased from clontech ( mountain view , ca ) and used at a final concentration of 500  ng / ml to induce ctr9-targeting shrna expression . dimethyl sulfoxide ( dmso ) and 17 -estradiol ( e2 ) were purchased from sigma ( st . louis , mo ) . in order to globally identify e2- and ctr9-regulated transcriptome in er breast cancer cells , mcf7-tet - on - shctr9 cells were maintained in dmem supplemented with 10% fbs in the absence or presence of 500  ng / ml dox for 4  days , followed by continuing culture in stripped medium with or without 500  ng / ml dox for another 3  days . cells were subsequently treated with dmso or 10  nm e2 for 4  h prior to cell collection ( fig . total rna from twelve samples was extracted using a qiagen rneasy plus kit according to manufacturer 's protocol . rna quality was assessed on the agilent 2100 bioanalyzer using a rna 6000 nano chip . 2 ) . 400  ng of total rna for each sample was used to synthesize double - stranded cdna and the corresponding in vitro transcription ( ivt ) crna , using the ambion wt expression kit per the manufacturer 's instructions . in order to monitor labeling and hybridization quality , subsequently , crna was purified and quantified and then subject to single strand ( ss ) cdna synthesis . the purified ss cdna was then fragmented to <  150  bp and end - terminus labeled using affymetrix genechip wt terminal labeling kit following manufacturer 's guidelines . the labeled samples were then hybridized to genechip human transcriptome array 2.0 containing >  6.0 million distinct probes covering 44,699 protein coding genes and 22,829 non - protein coding genes at 45  c for 16  h , according to the guidelines in the affymetrix genechip wt terminal labeling and hybridization user manual . after hybridization , the genechip arrays were washed and then stained ( streptavidin  phycoerythrin ) on an affymetrix fluidics station 450 followed by scanning on a genechip 3000 g7 scanner . data analysis and visualization were performed using the affymetrix transcriptome analysis console ( tac ) software per the manufacturer 's protocol . the criteria for selecting differentially expressed genes was 1.5-fold change cutoff and a false discovery rate <  0.05 . based on this criteria , we found that over 1600 genes were regulated by estrogen treatment , and of those 1600 , only 50 genes respond to e2 after ctr9 knockdown , suggesting the essential role of ctr9 in regulating er-dependent gene transcription . mcf7-tet - on - shctr9 cells were generated and characterized previously ,   and maintained in dmem supplemented with 10% fbs . doxycycline ( dox ) was purchased from clontech ( mountain view , ca ) and used at a final concentration of 500  ng / ml to induce ctr9-targeting shrna expression . dimethyl sulfoxide ( dmso ) and 17 -estradiol ( e2 ) were purchased from sigma ( st . louis , mo ) . in order to globally identify e2- and ctr9-regulated transcriptome in er breast cancer cells , mcf7-tet - on - shctr9 cells were maintained in dmem supplemented with 10% fbs in the absence or presence of 500  ng / ml dox for 4  days , followed by continuing culture in stripped medium with or without 500  ng / ml dox for another 3  days . cells were subsequently treated with dmso or 10  nm e2 for 4  h prior to cell collection ( fig . to ensure the biological confidence , three independent experiments were performed as illustrated in fig . 1 . total rna from twelve samples was extracted using a qiagen rneasy plus kit according to manufacturer 's protocol . rna quality was assessed on the agilent 2100 bioanalyzer using a rna 6000 nano chip . 2 ) . 400  ng of total rna for each sample was used to synthesize double - stranded cdna and the corresponding in vitro transcription ( ivt ) crna , using the ambion wt expression kit per the manufacturer 's instructions . in order to monitor labeling and hybridization quality , subsequently , crna was purified and quantified and then subject to single strand ( ss ) cdna synthesis . the purified ss cdna was then fragmented to <  150  bp and end - terminus labeled using affymetrix genechip wt terminal labeling kit following manufacturer 's guidelines . the labeled samples were then hybridized to genechip human transcriptome array 2.0 containing >  6.0 million distinct probes covering 44,699 protein coding genes and 22,829 non - protein coding genes at 45  c for 16  h , according to the guidelines in the affymetrix genechip wt terminal labeling and hybridization user manual . after hybridization , the genechip arrays were washed and then stained ( streptavidin  phycoerythrin ) on an affymetrix fluidics station 450 followed by scanning on a genechip 3000 g7 scanner . data analysis and visualization were performed using the affymetrix transcriptome analysis console ( tac ) software per the manufacturer 's protocol . the criteria for selecting differentially expressed genes was 1.5-fold change cutoff and a false discovery rate <  0.05 . based on this criteria , we found that over 1600 genes were regulated by estrogen treatment , and of those 1600 , only 50 genes respond to e2 after ctr9 knockdown , suggesting the essential role of ctr9 in regulating er-dependent gene transcription . we described here a dataset composed of microarray gene expression profiling of e2- and ctr9-regulated genes in mcf7 human breast cancer cells . with this genome - wide transcriptome analysis , we were able to demonstrate ctr9 , the key scaffold subunit in human pafc , as a master - regulator of estrogen signaling in er breast cancer . we believe that this dataset would provide valuable information for researchers who are interested in studying the estrogen response hierarchy in er breast cancer .