GSM8560131: BLA, APP/Tau, 9months, Trained, sample1; Mus musculus; RNA-Seq
Brains were mounted in a cutting chuck with Tissue-Tek® O.C.T. Compound before sectioning. Frozen coronal sections (300 μm) were obtained using a cryostat (Leica CM 3050s) and mounted on Superfrost Plus slides (J1800AMNT, Thermo Fisher Scientific). Next, hippocampi and BLA were obtained from eight and two consecutive slices (BLA: coronal plane from 1.3 to 1.9 mm behind Bregma), respectively. Both hippocampi were microdissected with a scalpel, and the BLA region was obtained using a 1 mm Ø diameter micropunch (BP-10F, Miltex kai Japan) (4 punches/mouse). Immediately after dissection, both hippocampi and amygdalae were placed individually in the ice-cold Dounce homogenizer (D8938-1SET, Sigma-Aldrich) vessel with a predetermined volume of pre-chilled homogenization buffer (50mM Tris pH 7.5, 100mM KCl, 12mM MgCl2, 1% Nonidet P-40, protease inhibitor, 1mM dl-Dithiothreitol (DTT), 1mg/ml heparin, 100ug/ml cycloheximide, 200U/ml RNAse Out in H2O RNAse free) to achieve a 2-10% w/v ratio; 750 μl of homogenization buffer were used for the hippocampus sample and 250 μl for the BLA sample. Both tissues were homogenized using 40 strokes with a large pestle (A) and 40 strokes more with a small pestle (B) to form the final homogenate. The lysate was centrifugated at 10,000 x g for 10 min at 4ºC to obtain a post-mitochondrial supernatant. A small aliquot of the cleared lysate (input) (100 μl from each tissue) was taken and stored at -80ºC for subsequent input analysis. cDNA libraries were prepared using the NEBNext® Poly(A) mRNA magnetic isolation module and NEBNext® Ultra™ II RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions. In the case of hippocampus samples, cDNA libraries were prepared from 50 ng of RNA whereas, for BLA samples cDNA libraries were prepared from 10 ng of RNA. Libraries  were sequenced on an Illumina NextSeq 2000 instrument with 40 million reads per sample. Each fragment was sequenced paired-end 2 x 50 bp.