title: GSM1832591: 6_Hour_Rep_3; Euphorbia esula; RNA-Seq
library_construction_protocol: RNA extraction was performed by grinding approximately 0.1 grams of frozen tissue in liquid nitrogen to a fine powder, and adding 1 ml Trizol reagent (Ambion/Life Technologies). Chloroform:Isoamyl (24:1) was added to the Trizol/tissue mixture, centrifuged, and RNA was extracted from the resulting supernatant using an RNeasy Plant Mini kit (Qiagen). Quality control was assessed using a nanodrop bioanalyzer on the RNA setting. Poly A RNA was extracted with the NEBNext Poly(A) mRNA Magnetic Issolation Module. cDNA libraries were created usining the NEBNext Ultra DirectionalRNA Library Prep Kit for Illumina. In short, 4000 ng of RNA was purified and fragmented into mRNA, followed by first and second strand cDNA synthesis. End repair and adenylation of 3’ ends preceded adapter ligation and PCR amplification. Library quality was assessed using an Agilent Bioanalyzer, and quantified for pooling by qRT-PCR using the PhiX Control Kit v2 according to manufacturer specifications. Libraries were paired-end sequenced on an Illumina HiSeq2000.