GSM6859076: Mecp2-null, replicate 1, scRNAseq; Mus musculus; RNA-Seq
One-month-old mice were euthanized by Avertin (2, 2, 2-Tribromoethanol, 20 mg/ml) overdose and transcardially perfused with 20 ml ice-cold phosphate-buffered saline (PBS). The mice were decapitated posterior to the occipital bone immediately, and removed the overlying skin and muscles from the skull. The dorsal part of the skull was carefully removed for isolating the dura. After peeling from the skullcap, the dura was cut into small pieces in ice-cold RPMI 1640 medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and incubated with an enzyme mix (0.25% Trypsin and 10 U ml−1 Collagenase type I) at 37 °C for 15 min. The solution was gently passed through 70 mm nylon mesh cell strainers with a sterile plastic plunger to yield a single-cell suspension. The single-cell suspension was then centrifuged at 300 g for 10 min. The supernatant was removed completely and the cell pellet was resuspended in FACS buffer (pH 7,4; 0.1 M PBS; 1 mM EDTA; 1% BSA). Magnetic separation of CD45 positive cells was performed according to the manufacturer's instructions for CD45 MicroBeads (Miltenyi Biotec, Bergisch Gladbach, Germany). RNA libraries were prepared with Chromiun Single cell 3' Reagent v3 Kits according to the manufacturer's protocol.