GSM5592769: dKO5; Mus musculus; RNA-Seq
Mice were anesthetized, perfused with saline and brain removed. Brains from 8 Wt, 8 Cnp-Cre:Jab1flfl mutant mice and 8 p21-/- Cnp-Cre:Jab1fl/fl double mutant mice at P40 were dissociated with 0.4 mg/ml of collagenase type IV (Sigma-Aldrich) and through a 18G syringe to obtain a homogeneous cell suspension. Cell suspensions were further enriched by an isotonic Percoll gradient, myelin was removed, cells washed in PBS and finally centrifuged and resuspended in MACS Buffer as described (Buttgereit et al., 2016).  Zombie Nir (BioLegend) was added to exclude non–viable cells. Samples were sorted on FACS Aria Fusion (BD Biosciences) and analyzed with Flow-jo (Three star). Mature oligodendrocytes were identified as single live cells CD45-O1+ and total RNA was isolated from sorted oligodendorcytes using ReliaPrepTM RNA Cell Miniprep System (Promega). Libraries were prepared using the SMART-Seq® v4 Ultra® Low Input RNA Kit for Sequencing (Takara Bio USA). Sequencing was performed on a Illumina NovaSeq 6000 machine (Illumina, San Diego, CA) obtaining an average of 100 millions of paired-end reads per sample.