GSM9497570: HC_148, AD; Homo sapiens; RNA-Seq
Dissected hippocampi were weighted to calculate the homogenization volume of the buffer for obtaining a 20% concentrated suspension (weight/volume). The tissue was homogenized in ice-cold 50 mM Tris–HCl (pH 7.4) supplemented with Halt™ Protease and Phosphatase Inhibitor Cocktail (#78438, Thermo Fisher Scientic, Pittsburgh, PA, United States) by using the Bio-Gen PRO200 Homogenizer (#01-01200, PRO Scientic Inc., Oxford, CT, United States) at setting three for 30 seconds and then at full speed for one minute. Subsequently, the suspension was centrifuged at 3000 x g for 10 minutes (4°C). Then, the supernatant was used for RNA extraction. Total RNA was isolated with RNeasy Mini Kit (#74104, Qiagen, Hilden, Germany), according to manufacturer's instructions to achieve maximum yields of RNA. Next, the samples were treated with the Heat&Run® gDNA Removal Kit (#80200, ArticZymes, Tromsø, Norway) to avoid amplication of genomic DNA during further processing steps. The SMARTer® Stranded Total RNA-Seq Kit v3 - Pico Input Mammalian (#634485, Takara Bio, Kusatsu, Japan) was used for library preparation, which was followed by a purication with AMPure XP beads (#A63880, Beckman Coulter Inc., Brea, CA, United States). Then, library fragments originating from rRNA (18S and 28S) and mitochondrial rRNA (m12S and m16S) were cleaved by ZapR v3 in the presence of mammalian specic R-Probes v3 (Takara Bio, Kusatsu, Japan). Paired-end sequencing for 50 bp each was performed on the NovaSeq 6000 platform (Illumina Inc., San Diego, CA, United States) to a depth of around 50 million reads (i.e., 25 million reads paired-end fragments).