title: Mesocricetus auratus organs mixture EST library
design: The eighteen total RNA samples were pooled equally into one pool. The total RNA pool underwent additional cleaning using the mirVana isoltation kit following manufacturer’s recommendations (Ambion). Poly A RNA cDNA was synthesized according to a standard protocol using an oligo(dT)-linker primer for first strand synthesis. The N0 cDNA was PCR amplified during 18 cycles using a high fidelity DNA polymerase. Normalization was carried out by one cycle of denaturation and reassociation of the cDNA, resulting in N1-cDNA. Reassociated ds-cDNA was separated from the remaining ss-can (normalized cDNA) by passing the mixture over a hydroxylapatite column. After hydroxylapatite chromatography, the ss-cDNA was amplified with 15 PCR cycles. For 454 sequencing, cDNA in the size range of 500-700 bps was eluted from a preparative agarose gel. An aliquot of the size fractionated cDNA was analyzed on a 1.5% agarose gel. 454 adaptors were ligated to the size fractionated N1 cDNA and 3' fragment sequenced on a Roche 454 using GS FLX technology with Titanium series chemistry following manufacturer’s recommendations.