GSM2802888: oldBPH2J.1; Mus musculus; RNA-Seq
Endothelium were acutely extracted from cardiac tissue using miltenyi microbeads (Cat# 130-097-418) according to the manufacore instructions. Beads were sorted on an autoMACS instrument using the POSSLD protocol. Positively sorted endothelial cells were spun down and disolved in 500uL of Qiazol and subsequently extracted using a RNAeasy universal mini RNA kit. PolyA(+) RNA is isolated using oligo-dT bound to magnetic beads.  Recovered RNA is chemically fragmented to about 200-300 bp.  Double stranded cDNA is made from the fragmented RNA using random hexamer priming.  The double stranded cDNA product is treated to “blunt” the ends.  A single “A” base is added to the ends of the fragments.  Proprietary Illumina adaptors are attached to the fragments by ligation. The unique “Y” structure of the adaptors ensures that different oligonucleotides are on the end of each strand.  The library is amplified by a limited number (usually 12) rounds of polymerase chain reaction.  The amplified library is cleaned using AMPure XP beads (Agencourt), then run on the Bioanalyzer (Agilent) to ensure that library preparation has been successful.  Sequenced 100 cycle single read on the HiSeq 2000.  Data was converted to fastq files with CASAVA (Illumina).
GEO
GSM2802888