GSM3214421: RNA-Seq CASE5; Mus musculus; RNA-Seq
Once the mouse is ready to be sacrificed for tissue extraction, the mice undergo a sacrifice and the head is cooled in liquid nitrogen. The brain is then dissected to isolate the hippocampus brain region and homogenize it in a lysis buffer. The homogenized hippocampus tissue is then kept on ice until all samples are ready to be processed for the next step. After homogenizing all hippocampi, the samples are spun at 2,000xg for 10min and supernatant removed. This supernatant is combined with detergents (NP-40 and DHPC) to help isolate polysomes. The samples are then spun again at 20,000g for 10min and supernatant removed. One aliquot of this supernatant is put aside to serve as a representative sample of total RNA for each homogenized hippocampus sample. The remaining supernatant is loaded onto a sucrose cushion (1M sucrose) and spun in an ultracentrifuge at 266,000xg for 4h. The resulting pellets were re-suspended in TRIzol and processed accordingly. Samples were then column-purified (Qiagen) to remove residual salts and genomic contamination. Next, RNA integrity was assessed using a BioAnalyzer (Agilent) at the MIT BioMicro Center. Following polysome enrichment, all RNA samples were prepared for RNA-seq using the SMARTer total RNA-seq kit (Clontech). The quality of prepared bar-coded libraries was assessed using a BioAnalyzer (Agilent) before mixing for sequencing on the Illumina NextSeq500 (Illumina) platform at the MIT BioMicro Center to generate 40bp paired-end fastq files generated from Illumina NextSeq500 platform.
GEO Accession
GSM3214421