title: GSM9054025: WT_S4_Brain; Mus musculus; RNA-Seq
library_construction_protocol: Processing and tissue collection were performed as previously described (69), using the Act-Seq method (70) to limit dissociation-induced gene expression. Briefly, the brains of 23-week-old homozygous mutated (Disc1LI/LI) and wild-type (Disc1WT/WT) littermate controls (n = 6 males per group) were pooled and cut into small pieces with a scalpel, then incubated with enzyme mix (30 U/ml DNAse I (Roche), 10 U/ml collagenase type I (Worthington) and 400 U/ml collagenase type IV (Worthington) diluted in 1X Hanks' buffered salt solution (HBSS, Gibco)), containing 15 μM ActD at 11 °C for 40 min. Every 10 min the solution was cut and resuspended to ensure full dissociation of the tissue. Subsequently, the solution was resuspended, filtered twice over a 100 μm nylon filter, and centrifuged at 400 g for 10 min. The pellet was resuspended in 5 ml 70% standard isotonic Percoll (SIP, GE Healthcare) diluted in 1X HBSS and gently overlaid with 5 ml of 30% SIP, followed by a 5 ml layer of 30% SIP, forming a three-layered density gradient (centrifuged at 650 g, 4°C, 30 min without acceleration/braking). All gradient buffers contained 3 µM ActD. The cell cloud at the 70/30% interphase was collected, centrifuged, and resuspended in staining buffer (2 mM EDTA, 2% FCS in 1X HBSS). Subsequently, cells were incubated with anti-mouse CD16/CD32 (clone 2.4G2), anti-CD45-Pacific Blue (clone 30-F11, BioLegend), and CD11bAF647 (clone M1/70, BioLegend) for 20 min on ice. CD45lowCD11b+ immune cells were sorted into 100% FCS using a BD FACS ARIA II with a sorting nozzle of 85 µm. Cellular suspensions were loaded on a Chromium Chip B (10 × Genomics, No.1000074), or Chip G (10 × Genomics, No. 2000177) on a GemCode Single Cell Instrument (10 × Genomics) to generate single-cell gel beads-in-emulsion (GEM). GEMs and scRNA-Seq libraries were prepared using the GemCode Single Cell 3' Gel Bead and Library Kit (v3 and v3.1, 10xGenomics, No. 1000075) and the Chromium i7 Multiplex Kit (10 × Genomics, No. 120262) according to the manufacturer's instructions.