Single-cell long-read sequencing of the experience-induced transcriptome

We performed single-cell long-read transcriptome profiling of mouse dorsal hippocampus following contextual fear conditioning, context exposure, or naive handling. Acute hippocampal slices from 16 C57BL/6J mice were dissociated, and single-cell suspensions were processed with the 10X Genomics 3 Gene Expression v3.1 kit. Barcoded cDNA was circularized and amplified with the R2C2 method, then sequenced on ONT PromethION (R9.4.1). Basecalling (Guppy v5.0.12) and consensus generation (C3POa + Racon v1.4.19) produced long reads demultiplexed by 10X barcodes and UMIs. Reads were aligned to the mouse GRCm39 genome and GENCODE vM32 transcriptome with Minimap2, and assigned to genes with FeatureCounts or isoforms with Salmon. After quality control, 20,000 cells across 21 hippocampal cell types were recovered, yielding gene- and isoform-level count matrices for downstream analysis