RNA editing in mitochondria of  Helianthus species
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.
SUB6429251
Helianthus_transcriptome_004