GSM1611867: control_RNAseq_1; Mus musculus; RNA-Seq
Hippocampi and right cortex were removed from all mice after sacrifice and immediately solubilized in QIAzol (QIAGEN).. Samples were homogenized by passing through a series of 3 increasingly higher-gauge needles. After homogenization, total RNA was isolated from the samples by following the protocol for the miRNeasy kit (QIAGEN). RNA was run on an Agilent 2100 Bioanalyzer to assess quality; only samples with excellent RNA quality (RNA Integrity Number [RIN] > 7) were used. Samples were depleted of DNA using Turbo DNase (Ambion). Poly-A- containing mRNA was selected from 20 µg of RNA using the Dynabeads mRNA direct kit (Invitrogen). The mRNA was fragmented, a 5’ phosphate was added using T4 polynucleotide kinase (Invitrogen), and the library was size- selected (60-200 nucleotides) on a 15% TBE-urea gel. Illumina v1.5 RNA adapters were ligated to ends of the fragmented products using T4 RNA ligase 2 (New England Biolabs). A cDNA library was generated using Superscript II (Invitrogen) and PCR-amplified using Phusion HiFi DNA polymerase (New England Biolabs). The library was purified on a 6% TBE gel (Invitrogen). The quality of the library was verified prior to Illumina sequencing by cloning into Zero Blunt TOPO (Invitrogen) and analyzing 6-10 clones by standard sequencing to determine a) the presence of adapters, b) appropriate size selection, c) the abundance of ribosomal RNA, and d) the diversity of the library products. The verified size-selected poly- A stranded cDNA library was submitted to the Penn Genome Frontiers Institute (PGFI, University of Pennsylvania, Philadelphia, PA) for sequencing. The Illumina Solexa GAII platform was used to obtain 50-bp single-end sequencing reads,
GEO Accession
GSM1611867