title: Transcriptome Analysis of ‘Suli’ Pear (Pyrus pyrifolia white pear group) Bud Dormancy by RNA-Seq
design: Briefly, after extracting the total RNA from the samples, mRNA is enriched by using the oligo(dT) magnetic beads 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 synthesize 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 are purified by agrose gel electrophoresis and enriched by PCR amplification. The library products are ready for sequencing analysis via Illumina HiSeq™ 2000.