Real time sequencer

mRNA was purified from 3 µg of mixed total RNA using poly (T) oligo-attached magnetic beads. Fragmentation was conducted using divalent cations under elevated temperatures in the NEBNext First Strand Synthesis Reaction Buffer (5×). The SMART PCR cDNA Synthesis Kit (Clontech, CA, USA) was used for synthesizing full-length cDNA. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of the 3′ ends of the DNA fragments, NEBNext Adaptor with a hairpin loop structure was ligated to prepare for hybridization. BluePippin® (Sage Science, Beverly, MA, USA) was used for size selection of the full-length cDNA and for building libraries of differently sized cDNA. The generated cDNA was then re-amplified using PCR, and the fragment size distribution was quantified using the Qubit fluorometer (Life Technologies, Carlsbad, CA, USA). The quality of the libraries was assessed using the Agilent Bioanalyzer 2100 system. SMRT sequencing was performed using the Pacific Biosciences’ real-time sequencer using C2 sequencing reagents.

After the RNA quality was verified, libraries were constructed. mRNA was purifed from 3μg of mixed total RNA of 21 samples of embryos of young loquat fruit for SMRT library preparation and sequencing. The instruments used include a SMARTer^™ PCR cDNA Synthesis Kit (Clontech, CA, USA)and BluePippin^® Size Selection System (Sage Science, Beverly, MA, USA). The SMARTer^™ PCR cDNA Synthesis Kit (Clontech, CA, USA) was used for synthesizing FL cDNA, the generated cDNAs were then reamplified via PCR. The remaining overhangs were converted to blunt ends by exonuclease/polymerase activities. After adenylation of the 3′ ends of the DNA fragments, NEBNext Adaptors with a hairpin loop structure were ligated in preparation for hybridization. The BluePippin^® Size Selection System was used for size selection(1–2 kb, 2–3 kb and 3–6 kb) to bulid 3 libraries.
The quality of the libraries was assessed using an Agilent Bioanalyzer 2100 system, and SMRT sequencing was performed using a Pacific Biosciences real-time sequencer in conjunction with C2 sequencing reagent.