GSM4508491: 8541_Brain_APPPS1PBS; Mus musculus; RNA-Seq
After manual perfusion, animals were decapitated and the brain region of interest was dissected from one brain hemisphere. Brain regions were immediately transferred to RNA later (Sigma) and stored at -80°C. Tissue was homogenized in 1 ml Trizol (TRI®Reagent; Sigma). For phase separation, 150 µl of 1 bromo 3 chloropropane (Sigma) were added, vortexed and centrifuged (15 min. at 12.000 × g at 4°C). After transferring the aqueous phase into a new tube 1 µl GlycoBlue™ (Invitrogen) and 500 µl 2-Propanol p.A. (Millipore) were added and vortexed. To obtain RNA, samples were centrifuged (10 min. at 12000 x g at 4°C). The pellet was washed with 1 ml 75% ethanol, dried and re suspended in 30 µl RNase-free water (pre warmed to 55°C). RNA was stored at -80°C for further analysis. The library preparation was done using TruSeq® Stranded mRNA Sample preparation kit (Illumina inc). The starting material (500 ng) of total RNA was mRNA enriched using the oligo(dT) bead system. The isolated mRNA was subsequently fragmented using enzymatic fragmentation. Then first strand synthesis and second strand synthesis were performed and the double stranded cDNA was purified (AMPure XP, Beckman Coulter). The cDNA was end repaired, 3' adenylated and Illumina sequencing adaptors ligated onto the fragments ends, and the library was purified (AMPure XP). The mRNA stranded libraries were pre-amplified with PCR and purified (AMPure XP). The libraries size distribution was validated and quality inspected on a Bioanalyzer 2100 or BioAnalyzer 4200 tapeStation (Agilent Technologies). High quality libraries are pooled based in equimolar concentrations based on the Bioanalyzer Smear Analysis tool (Agilent Technologies). The library pool(s) were quantified using qPCR and optimal concentration of the library pool used to generate the clusters on the surface of a flowcell before sequencing on a NextSeq500) instrument (75 cycles) according to the manufacturer instructions (Illumina Inc.).
GEO Accession
GSM4508491