title: 10nM+E2 Rep3
organism: Homo sapiens
description: Protocols: MCF-7 cells were purchased from ATCC and grown in standard culture conditions, these represent control cells. 300d-iAs MCF-7 cells were continuously exposed to sodium arsenite (iAs) at a concentration of 100nM for 10 months (300days). 16 day recovery MCF-7 cells were cultured in MCF-7 media without iAs for 16 days after the 10 month iAs treatment. Recovery cells were not re-exposed to iAs for the duration of the experiment. To determine the effect of iAs exposure to the estrogen response pathways, control cells and these iAs conditions were exposed to 10nM of 17β-estradiol (E2). Prior to E2 treatment, MCF-7, 300d-iAs MCF-7 and 16 day recovery MCF-7 cells were seeded at a density of 150,000 cell/well in 6 well plates in phenol red-free MEM media supplemented with 5% charcoal-stripped fetal bovine serum (i.e. treatment media). After 48 hours, the cells were treated with E2 (10 nM) for 24hr prior to RNA collection. Total RNA was isolated using RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. Total RNA (1ug) was aliquoted for library preparation utilizing Illumina's TruSeq Stranded mRNA protocol (catalog #20020594). Briefly, mRNA was captured by poly A magnetic beads, followed by fragmentation, cDNA synthesis, adenylation of fragments and ligation of adaptors for Illumina based sequencing.