title: GSM8189546: Pig 1, snRNAseq; Sus scrofa; RNA-Seq
library_construction_protocol: Nuclei were isolated from 80 mg thoracic and lumbar DRG tissue per sample employing previously published protocols (Ernst et al., 2020; Nguyen et al., 2021; Yang et al., 2021) that differ with respect to tissue lysis conditions (Triton X-100 or IGEPAL CA-630) and strategies employed for separation of debris and nuclei (40 µm filtration plus iodixanol density gradient centrifugation or 40 µm filtration plus magnetic-assisted enrichment of neurons using rabbit polyclonal anti-NeuN antibody (Millipore, cat# ABN78, dilution 1:5,000) and anti-rabbit IgG microbeads (Miltenyi Biotech, cat# 130-048-602, dilution 1:5)). The protocol by Ernst et al. that only relies on 40 µm filtration was modified to include an additional step of fluorescence-assisted enrichment of neuronal nuclei after the final two nuclei washing steps of the protocol. For this purpose, the nuclei were stained with Anti-NeuN-Alexa Fluor 555 (Millipore, cat# MAB377A5, dilution 1:100) for 30 minutes at 4°C on a rotator and subsequently with DAPI for 5 minutes on ice. At the FlowCore Mannheim, double-positive singlets were sorted into PBS/1% BSA using a BD FACSAria IIu equipped with a 100 µm nozzle. Single-nucleus solutions were subjected to barcoding, reverse transcription and gene expression library construction using the 10XGenomics microfluidics platform (Chromium controller and Next GEM Single Cell 3' Kit v3.1) according to the manufacturer's instructions.