GSM5139815: AD2659_KIF_18m: Hippocampal sample from App-NL-F mouse; Mus musculus; RNA-Seq
Total RNA was extracted from mouse hippocampal samples using a RNeasy Mini kit (Qiagen), according to the manufacturer's protocol and were sent for whole transcriptome sequencing at the Centro Nacional de Análisis Genómico (CNAG-CRG). Total RNA was assayed for quantity and quality using Qubit RNA BR Assay kit (Thermo Fisher Scientific) and RNA 6000 Nano Assay on a Bioanalyzer 2100 (Agilent). The RNA-Seq libraries were prepared from total RNA using KAPA Stranded mRNA-Seq Kit Illumina Platforms (Roche-Kapa Biosystems) with minor modifications. Briefly, after poly-A based mRNA enrichment with oligo-dT magnetic beads and 500ng of total RNA as the input material, the mRNA was fragmented (resulting RNA fragment size was 80-250nt, with the major peak at 130nt). The first strand cDNA was synthesized using random priming. The second strand cDNA synthesis was performed in the presence of dUTP instead of dTTP, to achieve the strand specificity. The blunt-ended double-stranded cDNA was 3'adenylated and Illumina indexed adapters (Illumina) were ligated. The ligation product was enriched with 15 PCR cycles and the final library was validated on an Agilent 2100 Bioanalyzer with the DNA 7500 assay. Each library was paired-end sequenced using TruSeq SBS Kit v4-HS, with a read length of 2x76bp. On average, we generated 53 million paired-end reads for each of the 48 samples of AppNL-F, AppNL-G-F, and WT mice in a fraction of a sequencing lane on HiSeq2000 (Illumina, Inc) following the manufacturer's protocol. Images analysis, base calling and quality scoring of the run were processed using the manufacturer's software Real Time Analysis (RTA 1.18.64) and followed by generation of FASTQ sequence files by CASAVA.