alias: GSM2311252
title: GSM2311252: Free-living myceliumG-2; Tulasnella calospora; RNA-Seq
library_construction_protocol: Total RNA was extracted  using the CTAB method. 100 mg aliquots  were mechanically  ground in liquid nitrogen and RNA was extracted in CTAB buffer (2% CTAB, 2%  PVP, 100 mM Tris-HCl pH 8.0; 25 mM EDTA pH 8; 2 M NaCl) at 65°C. 2% PVPP  was added to the buffer 1 h before the RNA extraction and 2% β-mercaptoethanol was  added to the buffer just before use. The homogenate was incubated at 65°C for 5 min  and extracted twice with chloroform: isoamylalcohol (24:1 v/v), each extraction  followed by 10 min centrifugation at 5000 rpm and room temperature. RNA was  precipitated overnight in 10 M LiCl at 4°C. After centrifugation at 10.000 rpm at 4°C  for 20 min, the pellet was dissolved in SSTE buffer (1 M NaCl, 0.5% SDS, 10 mM  Tris-HCl pH 8.0, 1 mM EDTA pH 8) and extracted with an equal volume of phenol (pH  4.5-5; Roti-Phenol, Roth A980): chloroform: isoamyl alcohol (25:24:1, v/v/v), followed  by chloroform: isoamyl alcohol (24:1, v/v). Each extraction step was followed by 10  min centrifugation at 10000 rpm and 4°C. Two volumes of 100% ethanol were then  added and RNA was precipitated for 2 hours at -20°C. After centrifugation for 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 1000 (Thermo Scientifics, USA) and Qubit 2.0 (Life  Technologies, Italy). RNA integrity was checked using a Bioanalyzer 2100 (Agilent  Technologies, Italy). cDNA libraries were prepared for sequencing using standard Illumina protocols by the Joint Genome Institue (JGI)
identifiers.namespace: GEO
identifiers.id: GSM2311252