Single- nucleus RNA Sequencing

The advance in single cell capturing and individual library construction technique combining with high-throughput sequencing allows gene expression studies on cell-by-cell basis. It enables a deeper and complete system analysis on complex cell populations, in which it largely avoids masking of their heterogeneity by taking average of all cells.

However, some cells are not suitable to be made into single-cell suspension, so other sample preparation methods – nucleus extraction from tissues are needed, that is, nucleus is directly extracted from tissues or cell and prepared into single-nucleus suspension for single-cell sequencing.

BMK provides 10× Genomics ChromiumTM based single-cell RNA sequencing service. This service has been widely used in studies on disease related studies, such as immune cell differentiation, tumor heterogeneity, tissue development, etc.

Spatial transcriptome chip: 10× Genomics

Platform: Illumina NovaSeq 6000

Service Details

Bioinformatics

Demo Results

Technical principle

The isolation of nucleus is achieved by 10× Genomics ChromiumTM, which consists eight-channel microfluidics system with double crossings. In this system, a gel beads with barcodes and primer, enzymes and a single nucleus are encapsulated in nanoliter-sized oil drop, generating Gel Bead-in-Emulsion (GEM). Once GEM are formed, cell lysis and release of barcodes are performed in each GEM. mRNA are reverse transcribed into cDNA molecules wih 10× barcodes and UMI, which are further subject to standard sequencing library construction.

Tissue not suitable for single cell suspension preparation

Cell / Tissue	Reason
Unfresh frozen tissue	Unable to get fresh or long-saved organizations
Muscle cell, Megakaryocyte, Fat…	Cell diameter is too large to enter the instrument
Liver…	Too fragile to break, unable to distinguish single cells
Neuron cell, Brain…	More sensitive, easy to stress, will change the sequencing results
Pancreas, Thyroid…	Rich in endogenous enzymes, affecting the production of single cell suspension

Single-nucleus vs Single-cell

Single-nucleus	Single-cell
Unlimited cell diameter	Cell diameter: 10-40 μm
The material can be frozen tissue	The material must be fresh tissue
Low stress of frozen cells	Enzyme treatment may cause cell stress reaction
No red blood cells need to be removed	Red blood cells need to be removed
Nuclear expresses bioinformation	The whole cell expresses bioinformation