GSM2775024: OX-29_rep1; Cucumis sativus; RNA-Seq
Total RNA samples were extracted from the young fruits of 4 days before anthesis (DBA) from 35S-CsFUL1 or wild-type cucumber plants, using the RNA extraction kit (Huayueyang, China). Approximately 3ug RNA per sample was used as input material for RNA-seq. The RNA-Seq libraries were constructed using NEBNext®Ultra™ RNA Library Prep Kit for Illumina® (NEB, Ispawich, USA) following manufacturer’s instructions, and four index codes were added to attribute sequences to each sample42. In brief, mRNA was purified using magnetic beads with Oligo (dT) (Life technologies, CA, USA), and fragmentation was carried out using divalent cations under elevated temperature in NEB proprietary fragmentation buffer. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). Second strand cDNA was subsequently synthesized using DNA Polymerase I and RNase H. After adenylation of 3’ ends of DNA fragments, NEBNext adaptor oligonucleotides were ligated to cDNA fragments, and AMPure XP beads system (Beckman Coulter, Beverly, USA) were used to select cDNA fragments of approximately 150~200 bp in length. DNA fragments with ligated adaptor molecules on both ends were selectively enriched using NEB Universal PCR Primer and Index primer in a 10 cycles PCR reaction. Products were purified (AMPure XP beads system) and quantified using the Agilent Bioanalyzer 2100 system. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) following the manufacturer’s instructions. After cluster generation, the libraries were sequenced on an Illumina Hiseq 2000 platform and 100 bp paired-end reads were generated.