title: GSM8410326: Sample8_HC; Homo sapiens; RNA-Seq
library_construction_protocol: The process of isolating nuclei involves breaking down cellular membranes using a detergent lysis technique, followed by centrifugal separation. Specifically, 300 mg of post-mortem brain tissue is homogenized in 2 ml of Nuclei EZ Prep Lysis Buffer with RNase inhibitor, DTT, and Triton X100. After a brief incubation on ice, the tissue is washed with PBSB, filtered, and combined with an ice-cold sucrose cushion solution. The mixture is centrifuged, and the supernatant and debris are discarded. The nuclei are resuspended in PBS buffer and a nuclei suspension buffer, then centrifuged again. DAPI is added to stain the nuclei, which are incubated and then resuspended in sorting buffer for quality inspection. Finally, fluorescence-activated cell sorting (FACS) is used to assess the quality, ensuring that over 95% of the events are DAPI-positive individual nuclei. Single-nuclei suspension concentration was determined by automatic cell counting (DeNovix CellDrop, DE, USA) using an AO/PI viability assay (DeNovix, DE, USA) and counting nuclei as dead cells. Single-nucleus gene expression libraries were generated using the 10x Chromium Next gel beads-in-emulsion (GEM) Single Cell 3' Reagent Kit v3.1 (10x Genomics, CA, USA) according to manufacturer's instructions. In brief, cells were loaded on the Chromium Next GEM Chip G, which was subsequently run on the Chromium Controller (10x Genomics, CA, USA) to partition cells into GEMs. Cell lysis and reverse transcription of poly-adenylated mRNA occurred within the GEMs and resulted in cDNA with GEM-specific barcodes and transcript-specific unique molecular identifiers (UMIs). After breaking the emulsion, cDNA was amplified by PCR, enzymatically fragmented, end-repaired, extended with 3′ A-overhangs, and ligated to adapters. P5 and P7 sequences, as well as sample indices (Chromium i7 Multiplex kit, 10x Genomics, CA, USA), were added during the final PCR amplification step. The fragment size of the final libraries was determined using the Bioanalyzer High-Sensitivity DNA Kit (Agilent, CA, USA). Library concentration was determined using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, MA, USA). snRNA libraries were pooled and paired-end-sequenced on the Illumina NovaSeq 6000 platform (Illumina, CA, USA).