GSM1529494: Syn_WT1_CT10; Mus musculus; RNA-Seq
In brief, mouse brains were homogenized in 5 ml homogenization buffer (0.32 M sucrose, 1 mM EDTA pH 7.4, 1 mM dithiothreitol, phenylmethanesulfonyl fluoride solution (Sigma, 93482-50ML-F), complete mini-protease inhibitor (Roche Diagnostics) for 10 sec using a polytron. The homogenate was centrifuged at 1,000g for 10 min at 4 °C yielding the nuclear fraction (Nuc) and the supernatant (Sup). The supernatant was centrifuged at 31,000g for 5 min at 4°C using a discontinuous Percoll gradient. The layer between 3% and 10% of Percoll were collected, washed in 30 ml of homogenization buffer and further centrifuged at 22,000 × g for 15 min at 4°CT. The pellet was resuspended in in EBC buffer (50 mM Tris-HCl pH 8.0, 120 mM NaCl and 0.5% NP-40) containing complete mini-protease inhibitor (Roche Diagnostics) and phosphatase inhibitor cocktail 1 and 2 (Sigma–Aldrich)) for Western blot analysis or lysis buffer for RNA extraction (GenElute Mammalian Total RNA Miniprep Kit, Sigma). For transcriptome analysis using RNA-Seq, the quality of the isolated RNA was determined with a Qubit® (1.0) Fluorometer (Life Technologies, California, USA) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). RNAs were then processed using The TruSeq Stranded mRNA Sample Prep Kit (Illumina, Inc, California, USA) according to the manufacturer recommendations. The TruSeq SR Cluster Kit v3-cBot-HS or TruSeq PE Cluster Kit v3-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on a Illumina HiSeq 2000 system using the TruSeq SBS Kit v3-HS (Illumina, Inc, California, USA) with paired end 2 X100 reads or single end 1X100 reads.
GEO Accession
GSM1529494