RNA-Seq for Phragmites australis: adult rhizome
Total RNA was extracted using Direct-zol RNA MiniPrep with TRIzol. DNA was removed by DNase I digestion added to the extraction column following the manufacturers protocol. RNA concentrations and qualities were estimated from 260/280 absorbance ratios via using a Nanodrop spectrophotometer. Due to the cost of sequencing at the time, samples did not have any biological and technical replicates. Paired-end RNA-seq library for each tissue was made using the ScriptSeq kit (Epicentre) following the manufacturers protocols with minor modifications. 5g of RNA were treated with Ribo-zero beads (Seed/Root beads are specific for rhizome and shoot meristem derived RNA; Leaf beads are specific for leaf and inflorescence derived RNA) to deplete ribosomal RNA. Approximately 50 ng of rRNA-depleted RNA from each tissue source was fragmented according to protocol with the exception that fragmentation at 85C was reduced from the recommended 5 minutes to 30 seconds. First-strand cDNA was made using random hexamers. This cDNA was terminally tagged and then amplified for 17 cycles using barcoded primers for each library based on tissue of origin.
@HWI-ST957:206:C1EHFACXX:3:1101:1204:2129