title: GSM5579302: ChIP_ECM_No_ECM_PD; Homo sapiens; ChIP-Seq
library_construction_protocol: RNA extraction: For gene expression analysis, total RNA was extracted from at least 3 biological replicates for each ligand treatment using Trizol reagent and further cleaned using the RNAeasy kit from QIAGEN.                           ChIP-seq analysis was performed as described previously using MCF7-ESR1Y537S cells. ChIP: ERα–DNA or IgG–DNA complexes were immunoprecipitated using ERα- specific F10 and HC20 antibodies (Santa Cruz Biotech, 3:100 dilution). Once the sample quality and replicate reproducibility was verified at least 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.The RNAseq libraries were prepared with Illumina's 'TruSeq Stranded RNAseq Sample Prep kit.  The libraries were quantitated by qPCR and sequenced on one lane for 101 cycles from each end of the fragments on a HiSeq2000  using a TruSeq SBS sequencing kit version 3. Fastq files were generated with the software Casava 1.8.2 (Illumina). 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.                                                                                          ChIP DNA was obtained from three pooled biological replicates. Libraries were prepared according to Illumina Solexa ChIP-Seq sample processing (San Diego, CA), and single-read sequencing was performed using the Illumina HiSeq 2000. Sequences generated were mapped uniquely onto the human genome (hg18) by Bowtie2 (RRID:SCR_016368). The MACS (model-based analysis of ChIP-seq) algorithm was used to identify enriched peak regions (default settings) with a P value cutoff of 6.0e−7 and FDR of 0.01, as we have described.