Transcriptome Analysis of ABA-Treated Embryos

Global transcriptome analysis was profiled using RNA isolated from embryos with or without ABA treatment, using a method similar to previously described^{63 (link)}. For each sample, equal quantities of high-quality RNA from 12 fruits were pooled for cDNA library construction. Briefly, 2 µg DNA-free total RNA from each sample were purified via oligo(dT)25 magnetic beads (Invitrogen), and then fragmented in 5 × first strand buffer (Invitrogen) at 94 °C for 2 min. The cleaved RNA fragments were reverse-transcribed into the first-strand cDNAs by 3′ and 5′ adaptors, followed by 18 cycles of PCR amplification with Illumina adapters with Phire II (Thermo Fisher Scientific). The library was run on an agarose gel, and a 300 to 500 nt band was excised and purified by using QIAquick gel extraction kit (Qiagen). The final PAT-seq libraries were quality checked by Qubit and Agilent Bioanalyzer 2100 before Illumina HiSeq 2500 sequencing was performed at an in-house facility at the College of the Environment and Ecology, Xiamen University. All RNA-seq read files are available from the NCBI Sequence Read Archive (SRA) (http://www.ncbi.nlm.nih.gov) (accession number SRP107989).