title: GSM4703684: A12; Dendrobium officinale; RNA-Seq
library_construction_protocol: Ethanol precipitation protocol and CTAB-PBIOZOL reagent was used to purify total RNA from PLBs and leaves according to the manual instructions, and each sample has three biological duplicates. 80 mg tissue samples were ground into powder in liquid nitrogen and the powdered samples were transferred into 1.5 mL of preheated 65℃ CTAB-pBIOZOL reagents. The sample solution was incubated by thermostatic mixer at 65°C for 15 min to completely dissociate the nucleoprotein complexes. The supernatant was obtained by centrifuging the solution at 12000 × g, at 4℃ for 5 min. The supernatant was mixed with 400 uL of chloroform (per 1.5 mL of added CTAB-pBIOZOL reagent), and the sample mixture was centrifuged at 12000 × g, at 4˚C for 10 min. The supernatant was transferred to a new 2 mL tube and added 700 μL of acidic phenol and 200 μL of chloroform into it followed by centrifugation at 12000 × g, at 4˚C for 10 min. The aqueous supernatant was collected and an equal volume of chloroform was added into it followed by centrifugation at 12000 × g, at 4℃ for 10 min. An equal volume of isopropyl alcohol was added into the supernatant and the mixture was placed at -20℃ for 2 h to precipitate before centrifuging it at 12000 × g, at 4℃ for 20 min, and the supernatant was removed. The RNA pellet was washed with 1mL of 75% ethanol before being air-dried in a biosafety cabinet and dissolved in 50 uL of DEPC-treated water. Subsequently, qualification and quantification of total RNA were performed using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). Oligo(dT)-attached magnetic beads were used to purify mRNA. The purified mRNA was split up into small pieces with fragment buffer at an appropriate temperature. The random hexamer-primed reverse transcription was performed to synthesize the first-strand cDNA followed by the second-strand cDNA synthesis. Then, A-Tail Mix and RNA Index Adapter were added to end the repair. The cDNA fragments obtained from above mentioned steps were amplified by PCR. The products were purified by Ampure XP Beads and dissolved in EB solution. The product was verified for quality control on an Agilent 2100 bioanalyzer. The double-stranded PCR products were denatured and cycled by the splint oligo-nucleotide sequence to obtain the final library, and the single-stranded circular DNA (ssCir DNA) was formatted into the final library. The established library was amplified with phi29 to obtain DNA nanoball (DNB), each molecular of which had over 300 copies. The DNBs were added into the patterned nanoarray and 100 base pairs of reads generated on the BGIseq500 platform.
attributes.tag: GEO Accession
attributes.value: GSM4703684