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, Germany). Integrity of purified RNA was
validated using a 2100 Bioanalyzer (Agilent, California, USA), and
concentration determined using a QuBit fluorometer (Thermo Fisher
Scientific, Massachusetts, USA). Plant rRNA was depleted from 2 µg of
purified RNA using the RiboZero rRNA removal kit for Plants (Illumina,
California, USA). Barcoded cDNA libraries were generated from our
rRNA-depleted RNA samples using the TruSeq RNA library preparation kit
(Illumina, California, USA). Sequencing of barcoded cDNA libraries was
performed at the Vincent J. Coates Genomics Sequencing Laboratory using
a HiSeq2500 platform using 50 bp single-end reads (Illumina, California,
USA).