Epigenomic Deconvolution

Normal Human Mammary Epithelial Cells (Clonetics), primary human fibroblasts (Asterand), human CD8+ cytotoxic T-cells (Sanguine), and breast cancer cell lines – MCF7, MDA-MB-231, MDA-MB-361, HCC1954, HCC1569, MCF10A (ATCC)—were cultured according to the respective manufacturer protocol. Logarithmically growing cultures were harvested at ~75-90% confluency. Frozen, primary human breast tumor tissue and adjacent normal tissue were obtained with local Institutional Review Board (IRB# PRO11090404) from the University of Pittsburgh’s Health Science Tissue Bank. Frozen samples were pulverized with mortar and pestle under liquid nitrogen conditions. DNA from cell culture, tumor tissue, normal tissue, and buffy coat was isolated using Qiagen’s DNeasy Blood and Tissue kit and bisulfite converted with the EpiTect Bisulfite Kit (Qiagen). Bisulfite converted DNA was quantified by Nanodrop, and for cell type mixture samples, mixed in the indicated proportions.

A set of 1000 target regions of around 300bp in length were preselected for targeted bisulfite sequencing. Primer pairs designed to specifically amplify each selected target region were designed by RainDance Technologies (TableS2.xlsx contains information about amplified regions and primer pairs used). The ThunderStorm BS-seq assay using that set of primer pairs was performed at RainDance Technologies according to the manufacturer’s specification. That assay uses a microfluidic chip to perform multiplex amplification of bisulfite treated DNA using the set of primers designed to amplify the selected set of genomic regions. This step is followed by sequencing of PCR product. Read mapping and methylation level calling was performed using Bismark. Target regions were sequenced on average to 200X coverage.