For gene expression analysis, total RNA was extracted from 3 biological replicates for each ligand treatment using Trizol reagent and further cleaned using the RNAeasy kit (QIAGEN). For time course studies, MCF-7 cells were treated with Veh (0.1% EtOH), 10 nM E2 or 1 μM PaPE for 4 h and 24 h. For inhibitor studies, MCF-7 cells were pretreated with Ctrl (0.1% DMSO), 1 μM PP242 or 1 μM AZD6244 for 30 min and then treated with Veh, 10 nM E2 or 1 μM PaPE-1 in the presence or absence of inhibitors for 4 h. Once the sample quality and replicate reproducibility were verified, 2 samples from each group were subjected to sequencing. RNA at a concentration of 100 ng/μL in nuclease-free water was used for library construction. cDNA libraries were prepared with the mRNA-TruSeq Kit (Illumina, Inc.). Briefly, the poly-A containing mRNA was purified from total RNA, RNA was fragmented, double-stranded cDNA was generated from fragmented RNA, and adapters were ligated to the ends.
The paired-end read data from the HiSeq 2000 were processed and analyzed through a series of steps. Base calling and de-multiplexing of samples within each lane were done with Casava 1.8.2. FASTQ files were trimmed using FASTQ Trimmer (version 1.0.0). TopHat2 (version 0.5) (55 (link)) was employed to map paired RNA-Seq reads to version hg19 of the Homo sapiens reference genome in the UCSC genome browser (56 (link)) in conjunction with the RefSeq genome reference annotation (57 (link)). Gene expression values (raw read counts) from BAM files were calculated using StrandNGS (version 2.1) Quantification tool. Partial reads were considered and option of detecting novel genes and exons was selected. Default parameters for finding novel exons and genes were specified. DESeq normalization algorithm using default values was selected. Differentially expressed genes were then determined by fold-change and p-value with Benjamini and Hochberg multiple test correction for each gene for each treatment relative to the vehicle control (9 (link)). We considered genes with fold-change >2 and Benjamini-Hochberg adjusted p-value less than or equal to 0.05 as statistically significant, differentially expressed. All RNA-Seq datasets have been deposited with the NCBI under GEO accession number GSE73663.
The paired-end read data from the HiSeq 2000 were processed and analyzed through a series of steps. Base calling and de-multiplexing of samples within each lane were done with Casava 1.8.2. FASTQ files were trimmed using FASTQ Trimmer (version 1.0.0). TopHat2 (version 0.5) (55 (link)) was employed to map paired RNA-Seq reads to version hg19 of the Homo sapiens reference genome in the UCSC genome browser (56 (link)) in conjunction with the RefSeq genome reference annotation (57 (link)). Gene expression values (raw read counts) from BAM files were calculated using StrandNGS (version 2.1) Quantification tool. Partial reads were considered and option of detecting novel genes and exons was selected. Default parameters for finding novel exons and genes were specified. DESeq normalization algorithm using default values was selected. Differentially expressed genes were then determined by fold-change and p-value with Benjamini and Hochberg multiple test correction for each gene for each treatment relative to the vehicle control (9 (link)). We considered genes with fold-change >2 and Benjamini-Hochberg adjusted p-value less than or equal to 0.05 as statistically significant, differentially expressed. All RNA-Seq datasets have been deposited with the NCBI under GEO accession number GSE73663.
