NET-Seq

Nuc-Seq

Nuc-Seq is an RNA sequencing technique optimized for isolating and sequencing nuclear RNA from frozen tissue samples. 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.

Pros:

• 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

Cons:

• 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

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

1. Jennings C. G., Landman R., Zhou Y., et al. Opportunities and challenges in modeling human brain disorders in transgenic primates. Nat Neurosci. 2016;19:1123-1130

1. 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