GSM3389640: Normal N3; Hylocereus polyrhizus; RNA-Seq
Tissues (100 mg) were placed into a precooled mortar to be ground into a powder with liquid nitrogen. The ground stem powder was collected into a precooled 2 mL Eppendorf tube with 2 mL RNase-free cetyltrimethylammonium bromide (CTAB) extracting solution, which contained 0.1% diethyl pyrocarbonate (DEPC), 2.5% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl (pH 8.0), and 4% polyvinylpyrrolidone (PVP). The tubes were then placed in a water bath at 65 °C for 30 min to induce cell lysis, and centrifuged at 13,000 rpm for 5 min at 25-28 °C. The residue was discarded, and the supernatant solution was transferred into a clean 2 mL Eppendorf tube. Trichloromethane (1 mL) was added to the supernatant solution, blended lightly, and centrifuged at 13 000 rpm for 15 min at 4 °C. The supernatant was transferred into a clean 2 mL Eppendorf tube, and this procedure was repeated once more. Subsequently, 8 M lithium chloride was added with RNase-free water to 0.6 volume of the supernatant and left overnight. The mixture was centrifuged for 30 min at 13 000 rpm and 4 °C to obtain the RNA precipitate. RNase-free ethyl alcohol (75%) was used to wash the RNA precipitate twice. Finally, RNA was dissolved in RNase-free ddH2O and stored at -80 °C until further use. Poly (A) mRNA from the total RNA was enriched by poly-T oligo-attached magnetic beads (Invitrogen), according to the manufacturer's protocol. Purified mRNA was then divided into short fragments by mixing with fragmentation buffer. The first strand cDNA was synthesized using a random hexamer primer. The buffer, dNTPs, RNase H, and DNA polymerase I were then added to the fragmented mRNA templates to synthesize the second-strand cDNA. The double-stranded cDNA was purified using magnetic beads. End repair and the addition of 3′-end single nucleotide A (adenine) were then performed. Finally, sequencing adaptors were ligated to the fragments, which were enriched by PCR amplification. During the quality control step, a 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA) and a StepOnePlus Real-Time PCR System (Applied Biosystems Inc., Foster City, CA, USA) were used to qualify and quantify sequences for the sample libraries. The constructed library products were prepared for sequencing using a HiSeq 2000 system (Illumina, San Diego, CA, USA) at the Beijing Genomics Institute (BGI).