title: Pisum sativum cultivar:Torsdag Raw sequence reads: Sample Control 1 hour
design: "RNA from pea node 2 axillary buds was extracted using a TRIzol extraction method and then purified using RNeasy MinElute cleanup kit (Qiagen).  RiboZeroTM Magnetic (Plant leaf) kit (Epicentre) was used to remove rRNA from 3.81 lg of each of the samples.  The removal of rRNA was confirmed using a 2100 BioAnalyser (Agilent Technologies).The RNA libraries were prepared using the ScriptSeqTM V2 RNA Seq Library Preparation kit (Epicentre), except for the following changes: the samples were incubated in the first step for 5 minutes at the lower temperature of 70 deg C to reduce RNA fragmentation, the cDNA was purified using the MinElute kit (Qiagen), and the ScriptSeqTM Index primers 1, 8, 9, 10, 11 and 12 (Epicentre) were used as adaptors.  Strand-specificity was not utilized. Size selection was performed by running the purified libraries on an agarose gel and excising a band of RNA between 350-550bps long.  This was then purified using the QIAquick Gel Extraction kit (Qiagen).  The QubitTM dsDNA HS Assay was used to quantify the RNA in each library. Four libraries were pooled for each sequencing run, with each library contributing 1.75 ng of RNA. The pooled libraries were prepared for sequencing using a MiSeq Desktop Sequencer (Illumina) and run on a 150 or 250 paired-end cycle cartridge."