GSM2522408: EMS_rep2; Chlorella vulgaris; RNA-Seq
Total RNA was extracted from 14-days old stationary phase reached (OD680=5) 5 ml cell culture using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions. Briefly, after quality and quantity measurement using 2100 BioAnalyzer (Agilent, USA), it was treated with RNase-free DNase I (Thermo Scientific, USA) at a concentration of 1 U/µg to remove residual genomic DNA. Then, RNA-seq library preparation was performed using TruSeq mRNA Sample Preparation Kit (Illumina, USA) according to the manufacturer’s instructions. mRNAs were purified from the 1 µg of total RNA using oligo (dT) magnetic beads and fragmented using fragmentation buffer. Afterthat, the cleaved short RNA fragments were used for first-strand cDNA synthesis using first strand synthesis mix, and the second strand was synthesized using second strand marking master mix. The double strand cDNAs were purified with AMPure XP beads (Beckman Coulter, USA) and eluted with resuspension buffer followed by 3’end adenine nucleotide addition. Finally, sequencing adaptors were ligated to the fragments and cDNA fragments were enriched by PCR amplification. Enriched cDNA libraries were used for cluster generation and sequencing. 75x2 paired-end sequencing of three cDNA libraries (WT, UV-715 and EMS-25) with two biological replicates were performed using the Illumina MiSeq sequencing platform (Illumina, USA). All sequence data are PE 2x75 bp.  Image processing, base calling, and quality calue calculation were performed by the Illumina data processing pipeline (v1.5). High quality reads were saved in FASTQ format.