GSM9335531: Donor_20_Dopa_neuron_Control; Homo sapiens; RNA-Seq
Nuclei were isolated from ventral midbrain tissue using Dounce homogenization in sucrose-based lysis buffer, followed by ultracentrifugation through a sucrose cushion to obtain purified nuclei. Immunotagging was performed using NeuN-Alexa488 and Nurr1-Alexa647 antibodies. Approximately 50,000 NeuN+/Nurr1+ (dopaminergic) and 50,000 NeuN-/Nurr1- nuclei were sorted on a BD FACSAria. RNA was extracted from sorted nuclei in TRIzol LS, followed by chloroform extraction and cleanup using the Zymo RNA Clean & Concentrator kit with extended DNase treatment. RNA-seq libraries were generated using the SMARTer Stranded Total RNA-Seq Kit v2 – Pico Input Mammalian (Takara). cDNA synthesis, rRNA depletion, adapter ligation, and PCR amplification were performed following the manufacturer's protocol. Libraries were sequenced on an Illumina NovaSeq-6000 (100 bp paired-end).