ECM Culture of Normal Breast Lines

We cultured htertHME, HME2, PCS600010, and MCF10A cells in decellularized ECM scaffolds generated by BJ fibroblasts or on an uncoated, plastic cell culture dish (Unc) to analyze the potential effects of ECM signalling breast cancer development. Total RNA was extracted from cells using TRIzol (Invitrogen) and purified using Direct-zol RNA mini kit (Zymo Research) with DNase I treatment. After RNA purification, samples were confirmed to have a RIN value > 9.0 when measured on an Agilent Bioanalyzer. Libraries for RNA-Seq were prepared with KAPA Stranded RNA-Seq Kit. The workflow consisted of mRNA enrichment, cDNA generation, end repair to generate blunt ends, A-tailing, adaptor ligation and 12 cycles of PCR amplification. Unique adaptors were used for each sample in order to multiplex samples into several lanes. Sequencing was performed on Illumina Hiseq 3000/4000 with a 150bp pair-end run. A data quality check was done on Illumina SAV. Demultiplexing was performed with Illumina Bcl2fastq2 v 2.17 program. Three replicate sets were generated by culturing htertHME, MCF10A, HME2, and PCS600010 cells in ECM scaffolds or on an uncoated surface in 6 cm dishes for 24 hours.