RNA-Seq of homo sapiens breast cancer cell line
Cultured cells were lysed and total RNA extracted (RNeasy Kit, Qiagen). mRNA was purified using poly-T oligo-attached magnetic beads, then fragmented randomly. cDNA was synthesized using random hexamer primers. Double-stranded cDNA was synthesized using DNA Polymerase I and RNase H then purified using AMPure XP beads. Remaining overhangs of the purified double-stranded cDNA were converted to blunt ends. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure was ligated to prepare for hybridization. After size-selection (250~300 bp inserts)  the final library is obtained by PCR amplification and purification of PCR products using AMPure XP beads.