The dynamic transcriptional profiling provides insights into the tuberous root development of Rehmannia glutinosa
To characterize the transcriptome of R. glutinosa during tuberous root formation and development, the experiment procedure in this study was designed as below. Firstly, a reference transcriptome was obtained by sequencing the mixed RNAs from three staged roots, i.e. adventitious roots (AR), thickening adventurous roots (TAR) and developing tuberous roots (DTR) using the illumina sequencing platform. Secondly, the single-ended reads were generated from cDNA libraries that were constructed from AR, TAR and DTR, respectively; then the gene expression profiling of three staged roots was obtained by mapping the reads to the reference, which gave the expression value of every gene by using RPKM method. The protocols used in this study were described as fellows. RNAs were firstly collected from three staged roots of R. glutinosa, and then poly(A) mRNAs were isolated using beads with Oligo(dT). Subsequently, fragmentation buffer was added for interrupting mRNA to short fragments. Taking these short fragments as templates, random hexamer-primer was used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNaseH and DNA polymerase I, respectively. Short fragments were purified with QiaQuick PCR extraction kit and resolved with EB buffer for end reparation and adding poly(A). After that, the short fragments were linked with sequencing adapters. After the agarose gel electrophoresis, the suitable fragments were selected for the PCR amplification as templates. At last, the libraries were sequenced using Illumina HiSeq™ 2000.