Genomic sample preparation kit

For Illumina transcriptome deep sequencing, total RNA was extracted from the fresh leaves of P. aphrodite subsp. formosana using the RNeasy Plant Mini Kit (Qiagen, Germany) according to the manufacturer’s protocol. RNA quantity and quality was verified using NanoDrop ND 1000 (Thermo Scientific, Hudson, NH, USA) and 2100 Bioanalyzer (Agilent Technologies), respectively. The cDNA library was constructed according to the manufacturer’s instructions for the mRNA-Seq Sample Preparation Kit (Illumina Inc., San Diego, CA). The constructed paired-end library was prepared by using the Genomic Sample Preparation Kit (Illumina) according to the manufacturer’s instructions. After validation on an Agilent Technologies 2100 Bioanalyzer, the library was sequenced using Illumina HiSeq^™ 2000 (Illumina Inc., San Diego, CA, USA) according to the manufacturer’s instructions. De novo transcriptome assembly for the high quality reads (Q < 20) was performed using Trinity software [79 (link)].

Isolation and enrichment of mRNA from total RNA was performed using oligo (dT) magnetic beads (Illumina, CA, USA). Then, mRNA was fragmented to short fragments to be used as templates for random hexamer-primed synthesis of first strand cDNA by fragmentation buffer. Second-strand cDNA was synthesized using buffer, dNTPs, RNase H, and DNA polymerase I. A paired-end cDNA library was synthesized using the Genomic Sample Preparation Kit (Illumina, CA, USA) according to the manufacturer’s instructions. Short fragments were purified with QIAQuick^®PCR extraction kit (QIAGEN, Germany) and eluted in 10 μL of EB buffer (QIAGEN, Germany). These short fragments were connected via sequencing adapters (Illumina, CA, USA). Agarose gel electrophoresis was used to select fragments approximately 50 bp in size. Finally, cDNA libraries were sequenced on an Illumina HiSeq^™ 2500 (Novogene, Beijing).

Isolation and enrichment of mRNA from total RNA was performed using oligo (dT) magnetic beads (Illumina, CA, USA). Then, mRNA was fragmented to short fragments to be used as templates for randomhexamer-primed synthesis of first strand cDNA by fragmentation buffer. Second-strand cDNA was synthesized using buffer, dNTPs, RNase H, and DNA polymerase I. A paired-end cDNA library was synthesized using the Genomic Sample Preparation Kit (Illumina,CA, USA) according to themanufacturer's instructions. Short fragments were purified with QIAQuick1 PCR extraction kit (QIAGEN, Germany) and eluted in 10 μL of EB buffer (QIAGEN, Germany). These short fragments were connected via sequencing adapters (Illumina, CA, USA). Agarose gel electrophoresis was used to select fragments approximately 50 bp in size. Finally, cDNA libraries were sequenced on an Illumina HiSeq™ 2500 (Novogene, Beijing).