Single- nuclei RNA Sequencing

Technical Scheme

The isolation of nuclei 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 with 10× barcodes and UMI, which are further subject to standard sequencing library construction.

Features

●   Preparation of single-nuclei suspension from frozen tissues

●   Formation of Gel Bead-in-Emulsion (GEM) followed by cDNA synthesis

●   Each bead in a GEM is loaded with primers composed of 4 sections:

                   poly(dT) tail for mRNA priming and cDNA synthesis,

                   Unique Molecular Identifier (UMI) to correct amplification bias

                   10x barcode

                   Binding sequence of partial read 1 sequencing primer

Advantages

Single-nucleus RNA sequencing circumvents the limitations of single-cell RNA sequencing, enabling:

● The use of frozen samples and not only limited to fresh samples

● Low stress of frozen cells when compared to enzymatic treatment of fresh cells, reflected in the transcriptome data in the form of less stress-induced genes

● No need for prior removal of red blood cells

● Unlimited cell diameter

● Large array of samples that are eligible for analysis, including complex and fragile tissue types that are prone to cell clumping or destruction during tissue dissociation

Samples that cannot be analysed by single-cell RNA sequencing and are eligible for single nuclei RNA sequencing:

Single-nucleus vs Single-cell

Specifications

For more details on sample preparation guidance and service workflow, please feel free to talk to a BMKGENE expert

Service Work Flow