GSM2104383: C_C_2; Cucumis sativus; RNA-Seq
Total RNA was isolated from each tissue using the TRIzol reagent according to the manufacturer’s protocol (Invitrogen, Burlington, ON, Canada). DNA was removed from RNA extracts by incubation with RNase-free DNase(New England Biolabs) for 30 min at 37°C. The RNA integrity and concentration was evaluated using an Agilent Technologies 2100 Bioanalyzer. Each sample had RNA Integrity Number (RIN) values greater than 7.5. Poly(A) mRNA was isolated from total RNA using oligo(dT) magnetic beads (Invitrogen, Carlsbad, CA, USA). Fragmentation buffer was added to break the purified mRNA into short fragments. Using these short fragments as templates, first-strand cDNA synthesis was performed using random hexamer primers and reverse transcriptase (Invitrogen). Second-strand cDNA was synthesized using RNase H (Invitrogen), DNA polymerase I (New England Biolabs), dNTPs and buffer. Subsequently, the short fragments were purified using the QIAquick PCR extraction kit, eluted with EB buffer and end repaired. Poly(A) tails were added, and the fragments were ligated to sequencing adaptors. We then selected suitable fragments as templates for PCR amplification according to the results of agarose gel electrophoresis. The average insert size for the paired-end libraries was 200 bp (from 180 to 220 bp). Sixteen paired-end cDNA libraries were constructed, one for each sample. Finally, the cDNA libraries were loaded onto the flow cell channels of an Uon Proton platform for paired-end 140 bp×2 sequencing at the Beijing Genomics Institute (BGI), Shenzhen, China.