title: GSM1474133: 409_rep2; Cucumis sativus; RNA-Seq
library_construction_protocol: Frozen fruit samples were grinded in a mortar with liquid nitrogen, and total RNA was isolated using RNA extraction kit (Huayueyang, China). RNA degradation and contamination was checked on 1% agarose gels, followed by Nano Photometer spectrophotometer (IMPLEN, CA, USA) to examine RNA purity. Qubit RNA Assay Kit in Qubit 2.0 Flurometer (Life Technologies, CA, USA) was used to measure RNA concentration, and RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA) was used to evaluate RNA integrity. Only RNA samples that passed the quality test were chosen for RNA-SEQ analyses. RNA-seq library construction was performed using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ispawich, USA) following manufacturer’s instruction and four index codes were added to attribute sequences to each sample45. Briefly, mRNA was enriched from 3ug total RNA using magnetic beads with Oligo (dT) (Life technologies, CA, USA), and then fragmented using divalent cations under elevated temperature in NEB proprietary fragmentation buffer. Double-stranded cDNAs were synthesized using random hexamers and M-MuLV Reverse Transcriptase (RNase H-), followed by 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 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) according to the manufacturer’s instructions. RNA-seq library were sequenced on an Illumina Hiseq 2000 platform and 100 bp single-end reads were generated.
attributes.tag: GEO Accession
attributes.value: GSM1474133