GSM9359390: C230215008 - PD2 - gene corrected; Homo sapiens; RNA-Seq
Frozen human brain organoids (18 samples), each tube containing approximately 30 organoids. All samples were handled on dry ice upon arrival. Nuclei were isolated following the manufacturer's instructions using the GEXSCOPE® Single Nucleus RNA Library Kit V2 (Singleron Biotechnologies; cat. nos. 1185011/2) and the included Nucleus Separation Solution V2. Briefly, tissue was minced on ice, incubated in Nucleus Separation Mix, mechanically dissociated, and filtered through a 40-µm strainer. The nuclei-containing flow-through was washed in PBS supplemented with EGTA, pelleted at low speed, and gently resuspended. Nuclear integrity and concentration were assessed microscopically using exclusion dyes such as DAPI or Trypan Blue. Sequencing libraries were generated using the GEXSCOPE® Single Nucleus RNA Library Kit V2 (cat. nos. 1185011/2). For all samples, 50 ng of amplified cDNA was used as input. cDNA was enzymatically fragmented, end-repaired, A-tailed, and ligated to truncated Illumina adapters. Adapter-ligated fragments were indexed by PCR using Unique Dual Indexes Set A (UDI Set A, included in the kit) and purified by dual size selection with AMPure XP beads. Library concentrations were quantified using a Qubit™ 4 Fluorometer (Thermo Fisher Scientific; cat. Q33238), and library fragment size distributions were evaluated using an Agilent TapeStation system (Agilent Technologies). Final libraries showed an average insert size of approximately 400–700 bp, with <5% of fragments below 300 bp, consistent with expectations for SCOPE-chip SD–derived single-nucleus RNA-seq libraries.