Towards a 3D spheroid system for modelling leukodystrophies 137 4 potassium ferricyanide and 1% aqueous osmium tetroxide in 0.1 M cacodylate buffer). Following the initial incubation with the heavy metal-based solutions, the pellets were washed with bi-distilled water at room temperature, then immersed in a 0.5% uranyl acetate solution and left overnight at 4 °C. The samples were dehydrated using a graded ethanol series (70%, 80%, 90%, 100%) and acetone for 10 minutes each, before being embedded in Epon resin. After curing at 60 °C for 48 hours, thin sections were cut using a Leica UC7 ultramicrotome (LeicaMicrosystems, Vienna, Austria). The sections were mounted onto 300-mesh copper grids and imaged using a Talos L120C G2transmission electron microscope (Thermo Fisher Scientific Inc., Waltham, MA, USA) at an acceleration voltage of 120 kV. Images were acquired at Advanced Light and Electron Microscopy BioImaging Centre of San Raffaele Scientific Institute (ALEMBIC) at IRCCS San Raffaele Scientific Institute. A Fei Talos L120C G2 transmission electron microscope was used. Image analysis and quantification were performed using ImageJ and Imaris software (NIH, Bethesda, MD). Bulk mRNA sequencing Total RNA was extracted from individual spheroids with the RNeasy Mini Kit (Qiagen, 74104) according to manufacturer protocol. Per donor, RNA of three spheroids were pooled to make one bulk mRNA sequencing sample. The RNA concentration and purity were determined using a NanoDrop spectrophotometer (Thermo Fisher Scientific). Purified RNA samples were sent to Genewiz (Azenta Life Sciences) for RNA sequencing (RNA-seq) analysis. Libraries were prepared with NEBnext Ultra II Directional RNA Library Preparation kit (New England Biolabs), and sequencing was performed on an Illumina NovaSeq 6000 platform. A minimum of 40 million read depth per sample was achieved. Quality control (QC) was conducted by the service provider to remove adapter sequences and low-quality reads using Trimmomatic v.0.36. Cleaned reads were aligned to the human reference genome GRCh38 available on ENSEMBL using the STAR aligner v.2.5.2b. BAM files were generated as a result of this step, unique gene hit counts were calculated by using featureCounts from the Subread package v.1.5.2. Single cell RNA-seq To obtain single cell transcriptomic data, three spheroids of each line at day 100 and 150 were dissociated. Brain spheroids were harvested from culture dishes and mechanically dissociated by mincing them into smaller pieces using a sterile scalpel blade (Bisturi) for 2– 3 minutes, until spheroids were visibly fragmented into fragments smaller than 1 mm in diameter. Care was taken to avoid excessive mechanical force to minimize cell damage.
RkJQdWJsaXNoZXIy MTk4NDMw