title: GSM9058830: 5 month old mouse brain hippocampus - WT genotype - sample 3; Mus musculus; RNA-Seq
library_name: GSM9058830
library_construction_protocol: mice were sacrificed via cervical dislocation at ~5 months of age. For transcriptomic analysis, both hippocampi were collected from a subset of randomly selected animals and stored at −80°C. Tissue homogenization was performed using the Precellys 24 Touch (Bertin Instruments, Montigny-le-Bretonneux, France) at 4500 rpm for 20 seconds. RNA extraction was carried out using the RNeasy Lipid Tissue Mini Kit (74804, Qiagen), following the manufacturer's instructions. RNA concentration was measured using the Qubit RNA Assay Kit (Thermo Fisher Scientific), and RNA integrity was assessed using the Agilent Bioanalyzer 2100 (Agilent Technologies, Les Ulis, France). Library preparation was performed using the Illumina Stranded mRNA Prep Ligation kit (Reference Guide – PN 1000000124518). RNA-Seq libraries were generated from 350 ng of total RNA following the manufacturer's instructions (Illumina Stranded mRNA Prep, Ligation kit and IDT for Illumina RNA UD Indexes, Illumina, San Diego, USA). After polyA selection, mRNA was fragmented at 94 °C for 8 minutes. DNA libraries were then amplified using 12 PCR cycles, and excess primers were removed by two rounds of purification using SPRIselect beads (Beckman-Coulter, Villepinte, France). Library quality and concentration were assessed using the Bioanalyzer 2100 system (Agilent Technologies, Les Ulis, France). Sequencing was performed on an Illumina NextSeq 2000 platform using single-end 50 base reads. Image analysis and base calling were conducted using RTA (v2.7.7) and BCL Convert (v3.8.4).