Nuc-Seq
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
Advantages:
- 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
Disadvantages:
- 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
Reagents:
Illumina Library prep and Array Kit Selector
Reviews:
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
References:
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
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History: Nuc-Seq
Revision by sbrumpton on 2017-06-21 07:50:22 - Show/Hide
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
Advantages:- 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
Disadvantages:- 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
Reagents:Illumina Library prep and Array Kit SelectorReviews:Jennings C. G., Landman R., Zhou Y., et al. Opportunities and challenges in modeling human brain disorders in transgenic primates. Nat Neurosci. 2016;19:1123-1130References: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