AB SOLiD System 3.0 sequencing; Breast Cancer Initiating Cells RNA-seq
Breast Cancer Initiating Cells RNA-seq
The MCF7 breast cancer cell line MCF7 was purchased from the American Type Culture Collection (ATCC; Manassas, VA). MCFS cells were derived in our laboratory from the MCF7 cell line (Ponti et al. 2005) and propagated as floating mammospheres. Cell vitality was assessed by Trypan Blue exclusion assay (at least 95%) before starting any experiment. Authentication of cell lines by STR DNA profiling analysis was performed at the A.O. Niguarda Ca Granda Hospital in Milan. Total RNA from 5000 MCF-7 and MCF-S cells was extracted following Agencourt RNAdvance Cell v2 (Beckman Coulter, MA, USA) protocol using Agencourts patented SPRI paramagnetic bead. Manufacturer instructions were followed with the following exception regarding DNase treatment step: Ambion TURBO DNA-free (Life Technologies technologiesCorporation, Carlsbad, CA, USA) was used instead of the one suggested in the kit. Ninety ng of total RNA was amplified, prior to transcriptome sequencing, using Ribo-SPIA technology developed by NuGEN following Ovation RNA-seq (2009, NuGEN Technologies, Inc.)  manufacturers instructions. The amplified cDNA was purified prior to subsequent processing for library construction using RNA Clean purification magnetic beads (Beckman Coulter Genomics, MA, USA) as suggested in the protocol. Double stranded DNA was quantified with Qubit ds DNA BR Assay Kits (Invitrogen, Ltd., UKLife technologies) and its size distribution was checked using the Agilent RNA Nano kit (Agilent Technologies, Inc.) following the manufacturers instructions.  DNA library, one for each sample, was constructed following the SOLiD 3 Plus System Library Preparation Guide (Life Technologies Corporation, Carlsbad, CA, USA) manufacturers instructions. Four emulsions PCR, two for each library, were manually performed following Applied Biosystems SOLiD 3 Plus System Templated Bead Preparation Guide, according to the manufacturers instructions (Life Technologies Corporation, Carlsbad, CA, USA). The libraries and emulsions quality were checked in a workflow analysis (WFA) run following SOLiD 3 Plus System Instrument Operation Guide (Life Technologies Corporation, Carlsbad, CA, USA).
GENOMNIA SRL
E-MTAB-2811:MCF7
Genomnia srl
E-MTAB-2811:MCF7
Experimental Factor: cell type
MCF7 epithelial cells
LIBRARY_STRAND
first strand