MiSeq sequencing library construction for identification of Rtsp-1 and LIb insertion sites
We constructed MiSeq sequencing library for identification of Rtsp-1 and LIb insertion sites in sweet potato. We designed the PCR primer at the specific region of these two kinds of retrotransposon families and amplified the junction sequence of retrotransposon and its insertion site. 
Genomic DNA was separated into fragments of approximately 6 kbp by g-TUBE (Covaris) centrifugation and purified using a QIAquick PCR Purification Kit (Qiagen). The purified products were treated with DNA polymerase I and T4 DNA polymerase to convert the heterogeneous ends via their physical fragmentation into blunt ends. The blunt ends were adenylated using a Klenow fragment. A forked adapter was ligated using T4 DNA ligase. The retrotransposon insertion sites were specifically amplified using nested PCR, where the first polymerase chain reaction (PCR) was performed with retrotransposon-specific (Rtsp-1_ppt or LIb_L_D4) and adapter-specific primer combinations using the ligated products as the template, and the second PCR was performed with primers of Rtsp-1 or LIb and an adapter sequence (AP3) using the initial PCR products as the template. The PCR products were selected by size (300???500 bp) using gel electrophoresis and purified with a QIAquick Gel Extraction Kit (Qiagen). The MiSeq sequencing adapters were ligated to the size-selected products from each cultivar using a TruSeq DNA-Sampling Preparation Kit (Illumina) to obtain cultivar-specific combinations of the dual index sequences in the adapters.(Rtsp-1: TTCCGCGGGCGCAACA, LIb: GACTTGGGTCCCTCTCTTTC)
OKAYAMA
DRX014092