Assay
Single-Nucleus RNA-seq: sanity checks and pitfalls
Nuclei are isolated from frozen tissue and profiled with the same droplet chemistry as cells, but roughly half of the signal is unspliced pre-mRNA, so counting must include introns or you lose most of the reads. Mitochondrial transcripts should be near absent, which flips the meaning of a mito filter, and ambient RNA from lysed cytoplasm is a larger fraction of each droplet than in whole-cell data. Cell-type proportions are also distorted relative to scRNA-seq, so comparisons across the two assays need care.
Who this is for: Neuroscientists, cardiac and kidney biologists, and anyone working from frozen or hard-to-dissociate tissue. They usually inherit scRNA-seq defaults and are surprised that intronic reads, low mitochondrial fraction and ambient contamination behave differently in nuclei.
- How to Detect Batch Effects in Single-Nucleus RNA-seq
Before you run Harmony on your frozen-tissue nuclei, find out whether the batch axis is technical noise or the condition you came to study.
- How to Find and Remove Doublets in Single-Nucleus RNA-seq
A cluster that co-expresses two lineage markers isn't always a transitional cell state, and the fix depends on telling homotypic from heterotypic doublets before you delete anything.
- How to Sanity-Check Marker Genes and Cell Type Labels in Single-Nucleus RNA-seq
FindMarkers will happily hand you a stress signature, an ambient RNA gradient, or a pseudoreplication artifact and let you name a cluster after it.
- How to Tell If You Overclustered in Single-Nucleus RNA-seq
Leiden will happily hand you fifty clusters from thirty cell types if the resolution lets it; the object itself can't tell you which cuts are real.
- How to Avoid Pseudoreplication in Single-Nucleus RNA-seq
5,000 nuclei from two donors is not 5,000 replicates: pick pseudobulk or a mixed model before you trust a single p-value out of FindMarkers.
- How to Choose Cell QC Thresholds in Single-Nucleus RNA-seq
Your snRNA-seq filter probably came from a whole-cell PBMC vignette, and in nuclei a high mito fraction means cytoplasmic contamination, not a dying cell.