GSM9286561: TDP-43Q331K cortex replicate 3; Mus musculus; RNA-Seq
Cortical tissue was dissected, wrapped in aluminum foil, flash-frozen in liquid nitrogen, and stored at −80 °C until processing. Total RNA was isolated by homogenizing tissue in 400 µL TRIzol (Thermo Fisher Scientific) with 80 µL chloroform and homogenizing beads, followed by centrifugation at 12,000 × g. The aqueous phase was transferred to a silica-column purification system (Quick-RNA MicroPrep Kit, Zymo Research) and processed according to the manufacturer's instructions. RNA was eluted and adjusted to a final concentration of 50 ng/µL for library construction. RNA sequencing libraries were generated at the University of Missouri Genomics Technology Core using the Illumina Stranded RNA Prep, Ligation Kit (Illumina, Cat. #20040534). mRNA was captured with oligo(dT) magnetic beads, fragmented, and used as template for first- and second-strand cDNA synthesis. Dual-indexed adapters were ligated, and libraries were PCR-amplified and purified using Axyprep Mag PCR Clean-up beads (Fisher Scientific). Library size distribution was assessed using the Fragment Analyzer, quantified with the Qubit HS dsDNA assay, and sequenced on an Illumina NovaSeq X.