alias: GSM3377509
title: GSM3377509: VmRoot_Cd2; Vaccinium myrtillus; RNA-Seq
library_construction_protocol: RNA was extracted O. maius-inoculated V. myrtillus roots  and from control roots 45 days after cultures started.   Total RNA was extracted using the CTAB method.  Aliquots (100 mg) of V. myrtillus roots were collected, frozen in liquid nitrogen, mechanically ground and RNA was extracted in warm (65°C) CTAB buffer (2% CTAB, 2% PVP, 100 mM Tris-HCl pH 8.0; 25 mM EDTA pH 8; 2 M NaCl). 2% PVPP was added to the buffer 1 h before performing the extraction letting it to get hydrated and 2% β-mercaptoethanol was added to the buffer just before use. The extraction suspension was incubated at 65°C for 5 min. Two chloroform/isoamylalcohol (24:1) extraction steps followed.  Each extraction was followed by 10 min centrifugation at 5000 rpm and room temperature. An overnight precipitation in 10 M LiCl at 4°C followed. After a centrifugation at 10.000 rpm 4°C for 20 min, pellet was dissolved in SSTE buffer (1 M NaCl, 0.5% SDS, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8). Extraction steps with an equal volume of 1:1 phenol (pH 4.5-5; Roti-Phenol, Roth A980):chloroform/isoamyl alcohol (24:1) and of chloroform/isoamyl alcohol (24:1) followed. Each extraction step was followed by 10 min centrifugation at 10000 rpm and 4°C. Two volumes of ethanol 100% were then added and a precipitation step of 2 hours at -20°C followed. After a centrifugation of 20 min at 10000 rpm and 4°C, the pellet was washed with 80% ethanol, centrifuged 10 min at 10000 rpm and 4°C, air dried on ice, re-suspended in DEPC-treated water and quantified using NanoDrop (Thermo Scientific) specrophotometer and bioanalyzer (Agilent). cDNA libraries were prepared for sequencing using standard Illumina protocols by the Joint Genome Institue (JGI)
identifiers.namespace: GEO
identifiers.id: GSM3377509