Single-Nuclei RNA Sequencing
snRNA-Seq uses a mild and quick nuclear dissociation protocol to isolate and sequence RNA within the nucleus. The method minimizes technical issues that can arise from common dissociation protocols, especially in studying immediate early gene (IEG) behavior (Lacar et al., 2016).
In this method, the cell suspension is lysed gently and the nuclei are separated from cytoplasmic lysates by centrifugation. Single cellsuclei are sorted into individual wells using FACS. Individual nuclei are amplified using a microfluidics-assisted machinery that performs cell capture and reaction chemistry. The nuclear RNA contents are processed into cDNA libraries using a Nextera XT DNA Library Prep Kit. Pools of 40 samples are collected and purified using magnetic beads. The cDNA library is ready for sequencing.
Similar methods: Div-Seq, Nuc-seq
- Rapid dissociation protocol prevents technical issues arising from protease digestion, heating, and spurious gene expression by cytoplasmic ribosomes
- Prevents dendritic loss that commonly occurs during protease dissociation step
- None reported yet
Illumina Library prep and Array Kit Selector
Poulin J. F., Tasic B., Hjerling-Leffler J., Trimarchi J. M. and Awatramani R. Disentangling neural cell diversity using single-cell transcriptomics. Nat Neurosci. 2016;19:1131-1141
Gagliano S. A. It’s All in the Brain: A Review of Available Functional Genomic Annotations. Biol Psychiatry. 2016;
Goncalves J. T., Schafer S. T. and Gage F. H. Adult Neurogenesis in the Hippocampus: From Stem Cells to Behavior. Cell. 2016;167:897-914
Lacar B., Linker S. B., Jaeger B. N., et al. Nuclear RNA-seq of single neurons reveals molecular signatures of activation. Nat Commun. 2016;7:11022