De novo sequencing and comprehensive analysis of purple sweetpotato (Impomoea batatas L.) transcriptome
Purple sweet potatoes were grown in the green house of Chinese Academy of Forestry. Fresh tuberous roots of purple sweet potatoes were harvested and were subsequently cleaned with sterilized water. The collected samples were then immediately frozen in liquid nitrogen and stored in a -80°C freezer for later use. According to the manufacturer’s protocol, the total RNA of the sample was extracted with BIOZOL total RNA extraction kit (Bioer Technology, Hangzhou, China). The RNA was qualified and quantified by a Nanodrop ND-1000 (Nanodrop technologies, Wilmington, DE, USA). A total of 10 µg of total RNA was used for Illumina RNA-seq.   According to the requirements of cDNA library construction for Illumina RNA sequencing, beads with oligo (dT) were used to isolate poly(A) containing mRNAs from the total RNA. Next, the mRNAs were broken into short fragments by adding fragment buffer. These short fragments were used as templates and a random hexamer was used as a primer to synthesize first-strand cDNA. Then,  second-strand cDNA was further synthesized using buffer, dNTPs, RNaseH, and DNA polymerase I. Short fragments were then purified with a QiaQuick PCR extraction kit (QIAGEN, USA) and resolved with EB buffer for end reparation and adding a poly(A) tail. Sequencing adapters were then connected to the short fragments. After agarose gel electrophoresis, suitable fragments (200±25bp) were isolated as templates for PCR amplification. Finally, the cDNA library was constructed for sequencing using Illumina HiSeq™ 2000.
The Research Institute of Forestry, Chinese Academy of Forestry
De novo root transcriptome sequencing of purple sweet potato