title: RNA-Seq of Tianma tuber
design: Total RNA of tuber tissue powders was extracted using an RNA kit (Thermo Fisher Scientific Inc, Waltham, MA, USA) following the guidelines offered by manufacturer. The concentration and quality of extracted total RNA were determined by Nanodrop 2000 (Thermo Fisher Scientific, USA). The integrity of the RNA samples was evaluated by agarose gel electrophoresis. The extracted total RNAs with concentrations  300ng/L, OD260/280 = 1.82.0, and good integrity of electrophoretic bands were used to construct the sequencing library as follows: (1) mRNA was extracted from total RNA using Oligo (dT) magnetic beads; (2) mRNA was broken into fragment sequences with about 300 bp length; (3) The first strand of cDNA was synthesized using the fragment mRNA as templates and random oligonucleotides as primers with the help of a M-MuLV reverse transcriptase system; (4) The RNA strand was degraded by RNaseH before synthesizing the second strand of cDNA with dNTPs with the help of a DNA polymerase I system; (5) Purified double stranded cDNA gel electrophoresis was performed and the end of the cDNA was repaired by adding an A-tail; (6) The sequencing connector was connected and the cDNA was screened for 250-300 bp lengths by Ampure XP beads; (7) PCR was used to amplify the screening cDNA and the PCR products were refined using Ampure XP beads again; and (8) the library was then obtained. More than 6G raw data per library was generated by a Hiseq 4000 sequencer (Illumina, San Diego, CA, USA).