GSM1608050: 130514-GBM2-L-LO; Mus musculus; RNA-Seq
Total RNA was isolated from snap-frozen flow sorted cell pellets using TRIzol-chloroform extraction. RNA was resuspended in 10µl Ambion Nuclease-free water (Life Technologies) and DNase treated (Ambion TURBO DNA-free Kit) For cDNA synthesis, we added 5µl of isolated RNA into the Ovation® RNA-Seq method.  500ng cDNA was fragmented using covaris shearing and processed for Illumina library construction with the Illumina paired-end LT indexing protocol as previously published (Mardis et al., 2009; Govindan et al., 2012). Each library was sequenced on the Illumina HiSeq, generating between 15-22Mbp per lane. RNA-Seq (300-500bp size fractionation): cDNA samples were constructed into Illumina libraries according to the manufacturer’s protocol (Illumina Inc, San Diego, CA) with the following modifications:  1) DNA was fragmented using Covaris S2 DNA Sonicitor using the following condtions: Duty Cycle: 5, Intensity: 4, Cycles/Burst: 200, Time: 90sec (Covaris, Inc. Woburn, MA).  Fragment sizes ranged between 100 and 500bp.  2) Illumina adapter-ligated DNA was amplified in a single 50ml PCR for five cycles.  3) Solid Phase Reversible Immobilization (SPRI) bead cleanup was used to purify the PCR and select for 300-500bp fragments.   We added 0.8 volumes of the AmpureXP solution (preformulated with polyethylene glycol and sodium chloride) to size select DNA molecules greater than 300 bp.  The size-fractioned cDNA was washed three times with 750 µl of 70% ethanol, the beads were dried, and the cDNA library was eluted off the beads by adding 20µl 10 mM Tris-HCl (pH 8.0).  The size-fractioned libraries were assayed using the Agilent BioAnalyzer High Sensitivity DNA chips.
GEO Accession
GSM1608050