Illumina HiSeq 2500 paired end sequencing; The ETS transcription factor ELF5 modulates estrogen action in breast cancer via participation in FOXA1-ER driven transcription.
The ETS transcription factor ELF5 modulates estrogen action in breast cancer via participation in FOXA1-ER driven transcription.
ELF5 Isoform 2 cell lines were created and maintained as described in Kalyuga et al. 2012. Cells were fixed in 1% formaldehyde, at 37°C for 10 min, washed 2× with cold PBS, scraped into 600 µl PBS with protease inhibitors (P8340, Sigma), spun 2 min at 6,000 g, washed as before, and snap frozen in liquid nitrogen. Puromycin (Sigma-Aldrich, St Louis, Missouri, USA) at 1ug/mL was used to maintain selection. Doxycycline (Dox, Sigma-Aldrich) at 0.1ug/mL in water was used daily to induce ELF5.  Four independent replicates for ER, FOXA1, ELF5-V5 and H3K4me3 ChIP-seq were performed according to the protocols described in published in “Chromatin Immunoprecipitation-Sequencing (ChIP-seq) for Mapping of Estrogen Receptor-Chromatin Interactions in Breast Cancer” by Holmes, Brown and Carroll 2016 (https://link.springer.com/protocol/10.1007%2F978-1-4939-3127-9_8), and our previous publications in Hurtado et al. 2011 and Kalyuga et al. 2012. DNA purification following IP was performed using phenol:chloroform:isoamyl alcohol and Phase Lock Gel tubes. Libraries were prepared using the TruSeq ChIP Sample Preparation Kit (A) from Illumina, with AMPure XP bead double-sided size selection.