GSM2064198: CT_24hT_rep2; Ulmus minor; RNA-Seq
Approximately 100mg of leaf tissue around the treated site was harvested and flash frozen in liquid nitrogen. Extraction of RNA from the ground material was performed in 15 mL Falcon tubes according to a phenol-based protocol, modified from Ikoma et al., 1996: In short, the material was extracted with 5mL homogenization buffer (0.2 M boric acid, 10mM EDTA and 2% SDS, adjusted to pH7.6 with Tris base, 5% ß-mercaptoethanol, 25mM dithiothreitol). The homogenization step was followed by three steps of phenol:chloroform:isoamyl alcohol (P/C/I; 25:24:1, v/v/v) extraction. In the second PCI step, ca. 0.05g of polyvinylpolypyrrolidone (PVPP) was added. After the third PCI step, clean-up of polysaccharides was achieved by separation with ethanol (20%), 560mM potassium acetate and one volume of chloroform:isoamyl alcohol (C/I, 49:1, v/v). This was followed by extraction with C/I (49:1, v/v) and overnight precipitation with lithium chloride (LiCl, 2.67M). After washing the pellet with 2M cold LiCl and thereafter in cold 80% ethanol, the pellet was dissolved in nuclease-free water, transferred to 2mL tubes and extracted again with one volume C/I and precipitated with LiCl (2.67M) overnight. Finally the pellet was washed in 2M LiCl, twice in 80% ethanol, dried and dissolved in nuclease free water. DNase digestion was performed using the TURBO DNA-free™ Kit (Ambion, Life Technologies, Carlsbad, CA, USA) according to manufacturer’s protocol. The RNA integrity and quality was analyzed with an Agilent 2100 Bioanalyzer on a Nano RNA 6000 chip (Agilent Technologies, Palo Alto, CA, USA). Illumina TruSeqRNA sample prep v2; polyA+ selection during library preparation; a gel-based size selection was used to obtain the final library
GEO Accession
GSM2064198