GSM5176004: CK 3h-3; Phyllostachys edulis; RNA-Seq
Total RNA of Moso bamboo was extracted using RNAprep Pure Plant Plus Kit (No. DP441; Polysaccharides & Polyphenolics-rich; Tiangen Co. Ltd, Beijing, China; http://www.tiangen.com/). A total amount of 1 μg RNA per sample was used as input material for the RNA sample preparations. Library construction was prepared using the TruSeq NEBNext UltraTM RNA Library Prep Kit, Illumina (NEB, USA). The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads and mRNA was fragmented using divalent cations in higher temperature in NEBNext First Strand Synthesis Reaction Buffer (5X). Using the random hexamer primer and M-MuLV Reverse Transcriptase, the first-strand cDNA was synthesized. Second strand cDNA synthesis was subsequently synthesized using DNA Polymerase I and RNase H. overhangs were converted into blunt ends using exonuclease activity. The NEBNext Adaptor containing a hairpin loop structure were ligated to prepare for hybridization. Further, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, USA) to select the cDNA fragments of 240 bp in length. Then 3 μl USER Enzyme (NEB, USA) was added to the adaptor-ligated, size-selected cDNA and incubated at 37°C for 15 min followed by denaturation 95°C for 5 min before PCR. Using Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer, PCR was performed. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.