title: GSM5980029: WT 4; Mus musculus; RNA-Seq
library_construction_protocol: After intracardiac perfusion, spinal cords from 10-12 month old KI and WT mice were dissociated in Hank's Balanced Salt Solution (HBSS) medium (Gibco), 1 mg/ml of DNAse I and 2 mg/ml of papain (Worhington) at 37°C for 30 min with mild shaking. Tissue suspensions were then filtered through a 70-mm cell strainer. Cell debris were eliminated after resuspension in HBSS 3.5% Bovine Serum Albumin (BSA, Sigma) and centrifuged at 120 g for 10 min. WT1, WT2, WT3, KI1, KI2 and KI3 were isolated as a single batch, while WT4 was isolated on a separate date. Cell pellets were resuspended in HBSS, layered over a 1.06 g/ml solution of Optiprep (Sigma). After centrifugation at 900g for 20 min, cells were collected in the upper layer, washed and resuspended in 1 ml of TRIzol. RNA from bulk spinal cord samples were isolated using Trizol, following manusfacturer's recomendations. RNA was DNAse-treated using the TURBO DNA-free Kit (Thermofisher scientific). 300ng RNA from the spinal cord samples was used for downstream library preparation. Libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB) following manufacturer's recommendations. Final libraries were quantified and sizing was determined using the High Sensitivity DNA Assay reagents and chip in the Agilent 2100 Bioanalyzer System and the Qubit 1X dsDNA HS Assay Kit respectively. Individual libraries were then pooled and sequenced using 75bp paired end on the NextSeq 500 using the NextSeq 500/550 High Output Kit.