SMRT sequencing of Chinese cabbage: anther
Total RNA from the three samples was extracted using Trizol reagent (Invitrogen, CA, USA). RNA purity and integrity was monitored by NanoPhotometer spectrophotometer (IMPLEN, CA, USA) and a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). RNA contamination was assessed by 1% agarose gel. RNA concentration was detected using a Qubit 2.0 Fluorometer (Life Technologies, CA, USA). Equimolar rations of the total RNA from each sample were mixed together. The full-length cDNA was prepared using a SMARTerTM PCR cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). Size fractionation (1-2, 2-3, and >3) of full-length cDNA was achieved using the BluePippinTM Size Selection System (Sage Science, Beverly, MA). The filtered full-length cDNAs were subjected to re-amplification, end repair, SMRT adapter ligation, and exonuclease digestion. After secondary screening by BluePippinTM, three SMRTbell libraries were constructed with the Pacific Biosicences DNA Template Prep Kit 2.0. Library quantification and size was measured using a Qubit 2.0 Fluorometer (Life Technologies, CA, USA) and Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Subsequently, SMRT sequencing was performed on a PacBio Sequel platform by Frasergen Bioinformatics Co., Ltd. (Wuhan, China).