Nanodrop 2000 micro spectrophotometer

Fresh leaves of H. myrtifolia (Lythraceae, Myrtales) were attained from Hangzhou Botanic Garden, Zhejiang Province (China), and were preserved immediately in silica gel. Genomic DNA was extracted employing a standard Cetyl trimethyl ammonium bromide (CTAB) protocol [40 ]. The concentration and quality of extracted DNA was evaluated using a NanoDrop 2000 Micro spectrophotometer and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
A sequence library was constructed using purified DNA following the manufacturer’s instructions. Using an Illumina HiSeq 2000 sequencer (Illumina Biotechnology company, San Diego, CA, USA), approximately 41,103,536 raw reads were obtained with paired-end (PE) 150 bp length reads.

Since 30d, 60d and 90d of warm stratification represented critical time points of seed dormancy release of A. tsaoko as reported in our previous study [18 ], the embryos of CK, S30, S60 and S90 were used in RNA extraction and transcriptome profiling analysis. To prepare Illumina RNA-seq libraries, the total RNA of 12 embryo samples from CK, S30, S60, and S90 (three replicates per dormancy release stage) were extracted using Takara MiniBEST Universal RNA Extraction Kit (Beijing, China) by the manufacturer’s instructions. The purity, concentration and integrity of each RNA sample were validated using 1% agarose electrophoresis, Nanodrop 2000 microspectrophotometer and Agilent 2100 Bioanalyzer, respectively. For RNA-seq libraries construction, RNA samples (RIN>7.5) were enriched by Oligo-dT magnetic beads. The purified RNA was fragmented into short pieces by fragment buffer and then the reverse transcription was carried out with N6 primer. The double-strand cDNA was end-repaired, adaptor-ligated, and followed by PCR amplification. The amplified PCR product was heat-denatured into single-stranded DNA, single-stranded DNA was then circularized to make a single-stranded circular DNA library. Finally, paired-end sequencing of the cDNA library was performed on the BGISEQ-500 platform.

The fresh leaves of six species of Lythraceae within Myrtales (D. grandiflora, T. natans, L. salicaria, L. inermis, W. fruticosa and R. rotundifolia) were obtained from the nursery of Zhejiang A&F University, and then immediately stored in silica gel. A CTAB method was used to extract the genomic DNA [52 (link)]. A NanoDrop 2000 Micro spectrophotometer and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) were employed to evaluate the concentration and quality of the extracted DNA. Following the manufacturer’s instructions, the purified DNA was used to build a sequencing library. The Illumina HiSeq 2000 sequencer (Illumina Biotechnology Company, San Diego, CA) was used to obtain paired-end (PE) reads of 150 bp [9 (link)].