GSM1462979: clone RY8-79; Hevea brasiliensis; RNA-Seq
Total RNA were isolated latex and leaf according to previously described (Tang et al., 2007). RNA integrity was confirmed using the 2100 Bioanalyzer (Agilent Technologies). Beads with Oligo(dT) were used to isolate poly(A) mRNA after total RNA (QIAGEN GmbH, Hilden, Germany). Following purification, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. Taking these short fragments as templates, the first-strand cDNA was synthesized by using random hexamer-primer and Superscrip (Invitrogen California, USA). The second-strand cDNA was synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments were purified with QiaQuick PCR extraction kit (QIAGEN GmbH, Hilden, Germany) and resolved with EB buffer for end reparation and adding poly(A). Then the short fragments were connected with sequencing adapters. After agarose gel electrophoresis, the suitable fragment was selected for the PCR amplification as templates.