A Single-Nucleus RNA-Seq from Frozen Tissues

Nuc-Seq is an RNA sequencing technique optimized for isolating and sequencing nuclear RNA from frozen tissue samples (Krishnaswami et al., 2016). Nuc-Seq avoids proteolytic treatment during nuclei dissociation from cells, minimizing gene expression changes resulting from common protease dissociation procedures; this step is similar to the one used in snRNA-seq. Next, Smart-seq2 is used for cDNA synthesis, increasing the full-length cDNA yield due to its template-switching mechanism. Finally, the sequencing library is prepared using Tn5 transposase tagmentation.

Similar methods: snRNA-seq, Div-Seq


  • Nonproteolytic nuclear dissociation minimizes gene expression changes during isolation of nuclei
  • Template-switching mechanism of Smart-seq2 during cDNA synthesis increases the yield of full-length cDNA strands
  • FACS isolation increases throughput
  • RNA from frozen nuclei gives better cDNA quality than cytoplasmic RNA
  • Can be used on frozen tissues


  • Excludes any information from cytoplasmic RNA
  • Low-copy transcripts are hard to detect due to low amounts of isolated nucleolar RNA
  • ncRNAs and other short non-poly(A) RNA species are challenging to detect


Illumina Library prep and Array Kit Selector


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Krishnaswami S. R., Grindberg R. V., Novotny M., Venepally P., Lacar B., et al. Using single nuclei for RNA-seq to capture the transcriptome of postmortem neurons. Nat Protoc. 2016;11:499-524