title: GSM8944107: FCDI_3; Homo sapiens; RNA-Seq
library_construction_protocol: Single-nucleus dissociation was performed following the 10x Genomics protocol with optimizations based on laboratory-specific conditions. RNA integrity of each sample was first assessed using an Agilent 2100 Bioanalyzer, and only samples meeting quality criteria were processed further. Brain tissue was sectioned into 1–2 mm³ pieces and transferred to a Dounce homogenizer containing lysis buffer (10 mM Tris-HCl, pH 7.4; 10 mM NaCl; 3 mM MgCl₂; 0.1% Tween-20; 0.1% IGEPAL; 2% BSA). The tissue was homogenized to ensure complete lysis and nuclear release. The homogenate was transferred to a centrifuge tube and spun at 4°C for 10 min. The supernatant was removed, and the nuclear pellet was resuspended in ice-cold wash buffer (1× PBS with 1% BSA and 0.2 U/μl RNase inhibitor) to maintain nuclear integrity. A 40 μm cell strainer was used to remove debris and large aggregates. Nuclear concentration and integrity were assessed using a hemocytometer and trypan blue staining. Myelin debris was removed using a myelin removal reagent, followed by additional centrifugation and washing steps to obtain purified nuclei. The final nuclear concentration was adjusted to 700–1200 nuclei/μl for downstream library preparation. Isolated nuclei were loaded into a 10x Genomics Chromium microfluidic chip and captured using the Chromium Controller. Nuclei were encapsulated with gel beads and reagents to form single-nucleus droplets (Gel Beads-in-Emulsions, GEMs). Within the GEMs, reverse transcription (RT) was performed to generate barcoded cDNA from nuclear mRNA. The cDNA fragments were then amplified via PCR. Library construction was completed using the 10x Genomics Single Cell 3' v3 reagent kit, followed by high-throughput sequencing on the Illumina NextSeq X Plus platform.