Gernera ray floret transcripome
The cDNA library preparation and sequencing reactions were conducted at the Beijing Genomics Institute (BGI) genomic centre, Shenzhen, China. Magnetic beads with Oligo(dT) were used to isolate the poly(A)+ mRNA after all of the total high quality 20 µg RNA samples were mixed. Fragmentation buffer was added in the presence of divalent cations at 94?for 5 min to interrupt the mRNA into short fragments of approximately 200 bp. These short fragments were used as templates, and random hexamer-primer was used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNaseH and DNA polymerase I. The short fragments were purified with a QiaQuick PCR extraction kit and resolved with EB buffer to end reparation and add poly(A)+. The suitable fragments (approximately 375 bp) with sequencing adaptors were selected as templates for the PCR amplification based on the agarose gel electrophoresis results. The samples were clustered in flow cells to construct the cDNA library and loaded onto an Illumina HiSeq™ 2000.