title: GSM6718809: A3; Dendrobium huoshanense; RNA-Seq
library_construction_protocol: 75μL EB（0.25M, NaCl; 0.05M, Tris-HCl (pH = 7.5); 20mM, EDTA; 1%(w/v) sodium dodecyl sulphate）, 750μL CI and 30μL β-mercaptoethanol were mixed in a 2.0mL RNase-free micro-centrifuge tube.Grinded tissue samples of the whole D. huoshanense seedlings at about 80 mg with liquid nitrogen into powder and transferred the milled powder samples to 2 mL tubes.  Vortex vigorously. Place at 20˚C for 5 minutes before centrifuging the samples at 4˚C for 5 min at 12000×g.1/10 volume of 3M sodium acetate (pH = 5.2) and 2.5 volume of absolute ethanol were added to the supernatant. Mix gently and incubate at 4˚C for 30 minutes.Centrifuge at 4˚C for 10 min at 12000×g to recover the nucleic acids.200 μL of DEPC-treated water was added to dissolve the nucleic acids and 500μL of 10M LiCl was added to the solution. Mix gently and then place on ice for 60 minutes.Centrifuge at 4˚C for 10 min at 12000×g. Discard the supernatant and wash the pellet with 800μL of 75% (V/V) ethanol. Air-dry in a ventilator for 5-10min.50 μL of DEPC-treated water was added to dissolve RNA.RNA integrity and quality were evaluated by 1% agarose gel electrophoresis and spectrophotometric analysis using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). Oligo(dT) attached magnetic beads were used to purified mRNA with eliminating rRNA and tRNA in the total RNA. Purified mRNA of control sample and cold treated samples of the whole D. huoshanense seedlings were fragmented into small pieces with fragment buffer at appropriate temperature. Then, First-strand cDNA was generated by random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. After that, RNA Index Adapters and A-Tailing Mix were added by incubating to end repair. The fragments of cDNA obtained from previous steps were amplified by PCR, and the PCR products were purified by Ampure XP Beads firstly, dissolved in EB solution. The products were validated on the Agilent Technologies 2100 bioanalyzer for quality control. The double stranded PCR products obtained from previous steps were heated to denature and circularized by the splint oligo sequence to get the final library, and the single stranded circular DNA (ssCir DNA) was formatted as the final library. The constructional final library was further amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of each molecular, DNBs were loaded into the patterned nano-array and single end of 50 bases reads were generated on MGI2000 platform (BGI-Shenzhen, China).