GSM5380943: PD747; Homo sapiens; RNA-Seq
For each individual, a tissue block of cortical grey matter from the anterior cingulate was sectioned at 80 µm thickness. Adjacent sections were subsequently used for bulk-tissue RNA isolation (2 sections per sample) or isolation of nuclei for single-nuclei RNA-sequencing. Nuclei were isolated using buffers prepared as in Krishnaswami et al., including nuclei isolation medium #1 (NIM1), nuclei isolation medium #2 (NIM2), Homogenisation Buffer (HB), 29% and 50% vol/vol iodixanol dilutions. Briefly, brain tissue sections were suspended in 800 µL HB and homogenised in a pre-cooled 2 mL dounce homogeniser, with five strokes of the loose pestle, followed by 10-15 strokes with the tight pestle. The homogenate was filtered through a BD Falcon tube with a cell strainer cap (35 µm) and centrifuged at 1000 g for 8 minutes. Thereafter, nuclei were subjected to an additional clean-up step (density gradient centrifugation), as detailed in Krishnaswami et al., albeit with centrifugation of the layered nuclei/29% iodixanol solution at 13,000 g for 40 minutes at 4°C. The supernatant was carefully removed, and the nuclei pellet washed with PBS buffer (PBS + 1% BSA + 0.2 U/ml RNAseIn), filtered through a BD Falcon tube with a cell strainer cap, centrifuged at 500 g for 5 minutes at 4 °C and washed again. Nuclei were counted using a LUNA-FL Dual Fluorescence Cell Counter (Logos Biosystems, L20001) using Acridine orange dye to stain nuclei. All samples were processed as per 10X Genomics Chromium Single Cell Reagent Kits Protocol, (chemistry: Single Cell 3' v2). Following manufacturer's guidelines, the samples were processed to target 10,000 nuclei per sample. Briefly, we performed 8 cycles of cDNA amplification and 14 cycles of final indexing PCR. cDNA concentrations were measured using Qubit dsDNA HS Assay Kit (ThermoFisher, Q32851), and cDNA and library preparations were assessed using the Bioanalyzer High-Sensitivity DNA Kit (Agilent, 5067-4627). All samples were pooled to equimolar concentration and sequenced together across twenty-eight lanes on an Illumina Hi-Seq 4000. cDNA and library preparations were assessed using the Bioanalyzer High-Sensitivity DNA Kit (Agilent, 5067-4627).