Illumina HiSeq 2000 paired end sequencing; RNA-seq of mRNA from MCF7 breast cancer cells and samples derived from normal breast tissues
RNA-seq of mRNA from MCF7 breast cancer cells and samples derived from normal breast tissues
MCF7 cells were cultured in RPMI1640 medium (GIBCO/BRL) supplemented with 10% (v/v) fetal bovine serum (GIBCO/BRL), 2.0 g/L sodium bicarbonate, and were incubated in a humidified 37C incubator with 5% CO2. MCF7 cells were grown to 85% confluence in 6 cm tissue culture dish. Each 6 cm dish was rinsed twice with 1X PBS. Total RNA was extracted using TRIreagent (Invitrogen) protocol. The integrity of the RNA extract was checked by 1.2% (w/v) agarose gel electrophoresis and the concentration of RNA was estimated by ultraviolet spectrophotometry. The normal breast tissue mRNA were purchased from Origene (Catalog No.: CR559104 and CR561898). The sequencing library for mRNA-seq was prepared using TruSeq RNA Sample Preparation Kit (Illumina Inc., USA) as per manufacturers instruction. Briefly, total RNA with RIN (RNA integrity number) greater than 7.5 was subjected for poly-A mRNA isolation using poly-T oligo-attached magnetic beads. The poly-A mRNA was fragmented and first-strand cDNA was synthesized using random hexamers following by second-strand cDNA synthesis, end repair, addition of a single A base and adapter ligation. The adapter-ligated cDNA library was sized-selected by agarose gel and amplified by PCR.
Experimental Factor: disease
normal