GSM5332561: Cerebral Cortex Female 5xFAD; Abi3 knockout - rep 4; Mus musculus; RNA-Seq
Total RNA was extracted from cortical tissues using TRI Reagent® (MRC). Briefly, mouse cortices were homogenized in TRI Reagent using a hand grinder. After 5 min incubation, chloroform was added onto the samples. Samples were centrifuged at 17,000g for 15 min at 4C. Top layer was transferred onto a new tube, processed with isopropanol for RNA precipitation. Samples were centrifuged and RNA pellets were washed with ethanol. RNA pellets were dissolved in nuclease free water and RNA concentration was determined using the Nanodrop 2000 Spectrophotometer (Thermo Fisher). Isolated RNA was shipped to Lexogen for library preparation and sequencing. Libraries were prepared manually according to the manufacturer's instructions using QuantSeq 3' mRNA-Seq FWD Library Prep Kit (Lexogen, UG Version 015UG009V0252). Briefly, 50 ng total RNA was denatured for 3 minutes at 85°C with oligo(dT) primers containing an Illumina-compatible sequence at its 5' end followed by reverse transcription at 42°C for 15 minutes. After RNA removal, the second strand of the cDNA was synthesized by a random primer containing an Illumina-compatible linker sequence at its 5' end, and the end product was purified by a magnetic bead-based method to remove all reaction components. Finally, individual sample barcodes (dual indexing) for multiplexing were introduced via PCR (cycle number determined by qPCR) and the leftover reaction components were removed by a magnetic bead-based purification method. All libraries were analyzed for adapter dimers, size distribution and concentration on a Fragment Analyzer System using the DNF-474 HS NGS Fragment kit (1-6,000 bp) (Agilent). After pooling the libraries in an equimolar ratio, the concentration and the size distribution of the lane mix was analyzed by Qubit dsDNA HS assay (Thermo Fisher Scientific) and by 2100 Bioanalyzer device using the HS-DNA assay (Agilent), respectively. A 2 nM dilution of the lane mix was denatured and diluted to loading concentration for sequencing on a NextSeq 500 instrument with SR75 High Output Kit (Illumina). Seventy-six-base-pair single-end reads were generated at Lexogen Services.