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).