alias: GSM2875090
title: GSM2875090: Free-living mycelium-3; Pezoloma ericae; RNA-Seq
library_construction_protocol: RNA was extracted from Rhizoscyphus ericae UMAH 7357 mycelia and from Rhizoscyphus ericae UMAH 7357-inoculated V. myrtillus roots 75 days after cultures start.  100 mg aliquots of mycelium were collected, frozen in liquid nitrogen, mechanically ground and RNA was extracted in Tris-HCl extraction buffer (100 mM Tris-HCl pH 8, 100 mM NaCl, 20 mM Na-EDTA, 0.1% PVP, 1% Na-laurylsarcosine sodium salt dissolved in DEPC-treated deionised water), mixed 1:1 with phenol (pH 4.5-5; Roti-Phenol, Roth A980). A phenol:chloroform:isoamyl alcohol (25:24:1) extraction step and a chloroform extraction step followed. Each extraction step was followed by 5 min centrifugation at 14000 rpm and 4°C. A isopropyl alcohol precipitation step at -80°C for 30 min was than performed followed by 30 min centrifugation at 14000 rpm and 4°C. The pellet was then re-suspended in a 1:1 DEPC treated water:6M LiCl solution and precipitated over-night at 4°C. After 30 min centrifugation at 14000 rpm and 4°C the RNA was rinsed with 70% ethanol, centrifuged for 5 min at 14000 rpm and 4°C, air dried on ice, re-suspended in DEPC-treated water and quantified using the nanodrop and the bioanalyzer.   Total RNA was extracted from V. myrtillus mycorrhizal roots using the CTAB method.  100 mg aliquots of V. myrtillus mycorrhizal 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 step was followed by 10 min centrifugation at 5000 rpm and room temperature. An overnight precipitation step 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 the nanodrop and the bioanalyzer." cDNA libraries were prepared for sequencing using standard Illumina protocols by the Joint Genome Institue (JGI)
identifiers.namespace: GEO
identifiers.id: GSM2875090
attributes.tag: GEO Accession
attributes.value: GSM2875090