2.5 ⎪ RNAseq library preparation and analysis
Two flash-frozen leaf discs of 0.73 cm2 collected at the end of the 15-h dark period were homogenized in liquid nitrogen by bead beating, and RNA was extracted and DNase-treated using the Qiagen RNeasy Plant Mini Kit (Qiagen, Hilden, Germany). Integrity of purified RNA was validated using a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and concentration determined using a QuBit fluorometer (Thermo Fisher Scientific, Massachusetts, USA). Plant rRNA was depleted from 2 mg of purified RNA using the RiboZero rRNA removal kit for plants (Illumina, San Diego, CA, USA). Barcoded cDNA libraries were generated from our rRNA-depleted RNA samples using the TruSeq RNA library preparation kit (Illumina, San Diego, CA, USA). Sequencing of barcoded cDNA libraries was performed at the Vincent J. Coates Genomics Sequencing Laboratory (Berkeley, CA, USA) using a HiSeq2500 platform with 50 bp single-end reads (Illumina, San Diego, CA, USA).