RNA editing in Helianthus species: Treated
Library preparation was performed using Illumina TruSeq RNA library     protocol developed by Illumina Technologies (San Diego, CA). One g of total RNA was     subjected to PolyA purification of mRNA. Purified mRNA was fragmented for 8 min at     elevated temperature (94 0C) in the presence of divalent cations and reverse     transcribed with SuperScript III Reverse Transcriptase by priming with random     hexamers. Second strand cDNA was synthesized in the presence of DNA polymerase I and     RNaseH. The cDNA was cleaned up using HighPrep PCR reagent (MAGBIO, Cat# AC-60050).     Illumina adapters were ligated to the cDNA molecules after end repair and addition     of A base. SPRI (solid-phase reversible immobilization, Beckman Coulter) cleanup was     performed after ligation. The library was amplified using 8 cycles of PCR for     enrichment of adapter ligated fragments. The prepared library was quantified using     Qubit and validated for quality by running an aliquot (1 l) on High Sensitivity DNA     Kit (Agilent). The effective sequencing insert size was ~130-380 bp; the inserts     were flanked by adapters whose combined size was ~130 bp. Transcriptome sequencing     was carried out with a Illumina- HiSeq system (Illumina, San Diego, CA) to obtain 80     million reads per sample.