GSM1525530: stalk_rep2; Cucumis sativus; RNA-Seq
Samples were fixed in Farmer’s Fixative, dehydrated through an ethanol/xylene series followed by a xylene/paraplast series, and embedded in paraplast as described previously17. Embedded samples were sectioned at 10-um-thick using a rotary microtome (YD-355AT Yidi Microsystems, China). The sections from pedicle, stalk and fruit were expanded in the Biological tissue spreading machine (KEDEE KD-P, China) with diethylpyrocarbonate-treated deionized water, and then mounted on RNAse free Leica FrameSlides. The slides with the sectioned samples were dried overnight at 42ºC and stored at 4ºC until used for laser microdissection. Laser microdissection was performed using Leica LMD 6500 (Leica Microsystems, Germany). Two biological replicates were performed for each tissue.Total RNA was extracted from the collected phloem tissues using the Arcturus PicoPure RNA isolation Kit (Applied Biosystems). DNaseI treatment was performed to remove potential DNA contamination using Qiagen DNaseI. TargetAmp 2-Round Aminoallyl-aRNA Amplification Kit 1.0 (Epicentre Biotechnologies) was used for RNA amplification according to the manufacture’s protocol. 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.