title: Development of polymorphic EST-SSR markers in Campeiostachys nutans using global transcriptome sequencing
design: For Illumina sequencing, the total RNA of each sample was isolated using the RNeasy Plant Mini Kit (Qiagen, Cat. # 74904). Both the quantity and quality of the RNA were verified using the NanoDrop ND1000 instrument (Thermo Scientific). A total of 20 µg of RNA was equally pooled from the 15 tissues for cDNA library preparation. The cDNA library was constructed following the manufacturer’s instructions of mRNA-Seq Sample Preparation Kit (Cat # RS-930-1001, Illumina Inc, San Diego, CA) (Illumina). Briefly, the total RNA was collected and pooled from nine C. nutans tissues, and magnetic oligo(dT) beads were used to isolate poly(A) mRNA. The RNA Fragmentation Kit (Ambion, Austin, TX) was used to cleave the mRNA into short fragments, which were then used as templates to transcribe into the first-strand cDNA using random hexamer-primer and reverse transcriptase (Invitrogen, Carlsbad, CA) (Invitrogen). Second-strand cDNA was synthesized using buffer, dNTPs, RNase H (Invitrogen), and DNA polymerase I (New England BioLabs, Ipswich, MA) (NEB). After that, a paired-end library was synthesized using the Genomic Sample Preparation Kit (Illumina), according to the manufacturer’s instructions. Short fragments were purified with the MinElute PCR Purification Kit (QIAGEN, Dusseldorf, Germany) (QIAGEN) and eluted in 10 µL of QIAGEN EB buffer end repair and the addition of poly (A). The short fragments were then connected with sequencing adapters, and desired fragments (200 ± 25 bp) were separated by agarose gel electrophoresis using a Gel Extraction Kit (Axygen Biosciences, Central Avenue Union City, CA). Finally, the sequencing library was built by PCR amplification (15 cycles) and sequenced using the Illumina Genome Analyzer IIx sequencing platform. Data analyses and base calling were performed by the Illumina instrument software.