RNA Extraction, Library Construction, and Sequencing
We extracted total RNA from the collected samples after sample
lyophilization using ReliaPrepTM RNA Tissue Miniprep System (Promega
Corporation) following manufacturer protocol. The quantity and quality
of the extracted RNA were assessed using a Qubit (Thermo Scientific),
while RNA integrity was confirmed by 1 % (w/v) agarose gel
electrophoresis and by a Bioanalyzer 2100 (Agilent Technologies) using
the RNA 6000 Nano LabChip Kit. RNA samples with RIN ≥ 6 were considered
suitable for sequencing.
Libraries for RNA sequencing were constructed using the TruSeq Stranded
mRNA Sample Prep LT Protocol (Illumina), and were sequenced with 2x100
bp paired-end reads on an Illumina HiSeq2500 platform, using a HiSeq
Flow Cell v4 with HiSeq SBS Kit v4 (Illumina) at the Centro de Genômica
Funcional Aplicada à Agropecuária e Agroenergia, ESALQ, USP, Brazil.