Single Molecule Real-Time  sequencing of  sugarcane : root,leaf,stem
One microgram of total RNA per reaction per tissue was reverse transcribed using the Clontech SMARTer cDNA synthesis kit and tissue-specific barcoded oligo dT in separate PCR tubes, to generate barcoded FL cDNA. Two or three RT reactions per tissue were run in parallel. PCR optimization was used to determine the optimal amplification cycle number for the downstream large-scale PCR reactions. A single primer (primer IIA from the Clontech SMARTer kit 5- AAG CAG TGG TAT CAA CGC AGA GTA C -3) was used for all PCR reactions following RT.Large-scale PCR products were purified with AMPure PB beads and quality control (QC) was performed on a 2100 BioAnalyzer (Agilent). Equimolar ratios of six cDNA libraries were pooled together. A total of 3.8 g cDNA was subjected to size fractionation using the SageELF system. Size fractions eluted from the run were subjected QC and pooled in equimolar ratios for subsequent re-amplification to yield six libraries (<1, 12, 23, 35, 46 and >5 kb). The pooled PCR products were purified using AMPure PB beads.One to five micrograms of purified amplicons were subjected to Iso-Seq SMRTBell library preparation (https://pacbio.secure.force.com/SamplePrep) Three barcoded SMRTBell libraries (35, 46 and >5 kb) were size-selected using the Sage BluePippin system to remove trace amounts of small inserts. A total of 47 SMRT cells were sequenced on the PacBio RS II platform using P6-C4 chemistry with 34 h movies.