Total RNA was extracted separately from DOL and NDOL using Trizol Reagent following manufacturer’s instructions. Approximately 10μg RNA from each sample was used to construct individual DGE libraries. After extracting the total RNA from the samples, mRNA of eukaryotes is enriched by using the oligo(dT) magnetic beads and mRNA of prokaryotes is enriched just by removing rRNAs from the total RNA. Adding the fragmentation buffer, the mRNA is interrupted to short fragments (about 200 bp), then the first strand cDNA is synthesized by random hexamer-primer using the mRNA fragments as templates. Buffer, dNTPs, RNase H and DNA polymerase I are added to sythsize the second strand. The double strand cDNA is purified with QiaQuick PCR extraction kit and washed with EB buffer for end repair and single nucleotide A (adenine) addition. Finally, sequencing adaptors are ligated to the fragments. The required fragments is purified by agrose gel electrophoresis and enriched by PCR amplification. The library products are ready for sequencing analysis via Illumina HiSeq™ 2000.
GEO Accession
GSM1171490