Abstracts

Rationale: Our recent work has shown that hyperexcitability exists from the earliest stages of disease progression (1-3 month-old) in the Tg2576 mouse model of Alzheimer’s disease (AD), which exhibit interictal spikes (IIS) and seizures. This early hyperexcitability accompanies molecular and functional alterations across many cell types in the hippocampal dentate gyrus (DG), including granule cells (GCs) and hilar mossy cells (MCs) which we have shown to be hyperexcitable in young Tg2576 mice. However, the full transcriptional ecosystem of early alterations across multiple DG cell types that may lead to hyperexcitability is unknown. To investigate this question, we developed a single-nucleus RNA sequencing protocol that can be readily followed by non-specialists in genomics. Our protocol uses Particle-templated Instant Partition (PIP) sequencing because PIP-seq enables single nuclei to be encapsulated in an emulsified droplet with RNAase inhibitors and barcodes. Once encapsulated, PIP samples are stable at room temperature for 72 hours and can be transferred to an off-site core facility for cDNA library preparation and sequencing. Initial pilots using our “RNA sequencing for Dummies” protocol detected putative cell type-specific alterations of epilepsy-related genes in the DG of young Tg2576 mice.

Methods: Tissue was obtained by quickly extracting the brain after intracardial perfusion with a high sucrose artificial cerebrospinal fluid protecting solution (aCSF). DG tissue samples were collected from horizontal brain slices of 500 µm using tissue punches of 1 mm diameter and were flash-frozen on dry ice. Nuclei were extracted using a 10X Genomics Chromium kit according to modified instructions that allow this nuclei extraction method to be compatible with Fluent PIP-seq T2 kit components. PIP encapsulation was performed on-site according to Fluent kit instructions. Library preparation and Illumina RNA sequencing were performed by the NYU Genome Technology Center according to Fluent kit instructions. Analysis included PIPseeker, Seurat 5, and functional characterization software, all freely available.


Results: We present a user-friendly protocol for non-specialists in genomics to perform PIP-sequencing studies and an analysis workflow that combines validated free tools to allow beginners to analyze PIP-sequencing datasets. Initial pilots using our “RNA sequencing for Dummies” pipeline have already identified putative transcriptional alterations in epilepsy-related genes in nuclei of GCs and MCs from 1 month-old Tg2576 mice (vs wild-type littermates).


Conclusions: “RNA sequencing for Dummies” is a user-friendly single-nucleus transcriptomics protocol that can reveal molecular alterations in specific DG cell types relevant to epileptogenesis. Our work will broaden access to advanced DG transcriptomic profiling and will reveal cell type-specific alterations that may underlie hyperexcitability in early AD.


Funding: This project was supported by the NIH R01 AG055328 to HES, Alzheimer’s Association grant AARFD-22-926807 to DAG and HES and by NIH T32 AG052909 to GSS and HES.