GSM1187661: TCM9450; Mus musculus; RNA-Seq
Flash-frozen tissues were homogenized and RNA extracted with TRIzol Reagent and purified with RNeasy columns (Qiagen), all samples with RINs greater than 7. Oligo d(T)25 Magnetic Beads (New England BioLabs) were used to isolate mRNA from 1-3μg of total RNA. Purified mRNA was fragmented with Ambion's RNA fragmentation kit for 5 minutes at 70°C. Library construction was adapted from the dUTP strand-specific protocol of Levin et al. 2010. mRNA was ethanol precipitated, concentrated in 5μl of water, and pre-mixed with 3μg of random hexamers for 5 minutes at 65°C before chilling on ice. First-strand cDNA was synthesized as described, but incubated as follows: 10 minutes at 25°C, 50 minutes at 42°C, and 15 minutes at 70°C. First-strand cDNA was purified by PCI extraction, ethanol precipitated with 0.1 volume 3M ammonium acetate to remove dNTPs, and resuspended in 104μl H2O. Second-strand cDNA was synthesized as described, but incubated for 2.5 hours. Paired-end libraries for Illumina sequencing were then prepared from the cDNA as previously published except adaptor-ligated cDNA was size-selected to 200-400bp, and we performed PCR using Phusion High-Fidelity DNA polymerase with HF buffer (New England BioLabs) and 10ul of Q Solution (Qiagen). These paired-end, strand-specific cDNA libraries were then sequenced on the Illumina Genome Analzyer (36 bp reads) or HiSeq (40 bp reads).
GEO Accession
GSM1187661