The development of single-cell capture and custom library construction techniques, coupled with high-throughput sequencing, has revolutionized gene expression studies at the cell level. This breakthrough allows for deeper and more comprehensive analysis of complex cell populations, overcoming the limitations associated with averaging gene expression over all cells and preserving the true heterogeneity within these populations. While single-cell RNA sequencing (scRNA-seq) has undeniable advantages, it encounters challenges in certain tissues where the creation of a single-cell suspension proves difficult and requires fresh samples. At BMKGene, we address this hurdle by offering single-nucleus RNA sequencing (snRNA-seq) using the state-of-the-art 10X Genomics Chromium technology. This approach broadens the spectrum of samples amenable to transcriptome analysis at the single-cell level.

The isolation of nuclei is accomplished through the innovative 10X Genomics Chromium chip, featuring an eight-channel microfluidics system with double crossings. Within this system, gel beads incorporating barcodes, primers, enzymes, and a single nucleus are encapsulated in nanoliter-sized oil drops, forming Gel Bead-in-Emulsion (GEM). Following GEM formation, cell lysis and barcode release occur within each GEM. Subsequently, mRNA molecules undergo reverse transcription into cDNAs, incorporating 10X barcodes and Unique Molecular Identifiers (UMIs). These cDNAs are then subjected to standard sequencing library construction, facilitating a robust and comprehensive exploration of gene expression profiles at the single-cell level.

Platform: 10× Genomics Chromium and Illumina NovaSeq Platform

  Technical Scheme

The isolation of nuclei is achieved by 10× Genomics Chromium™, 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

  Service Work Flow

Includes the following analysis:

- Quality Control : number of cells, gene detection, accurate identification of cells, RNA molecules and expression quantification

- Inner Sample Analysis:

Cell clustering and cluster annotation
Differential Expression Analysis: identification of DEGs in clusters
Functional annotation and enrichment of cluster DEGs

- Inter-group Analysis:

- Combination of data
- Differential Expression Analysis: identification of DEGs in groups
- Functional annotation and enrichment of  group DEGs

- Advanced Analysis:

- Cell cycle analysis
- Pseudotime analysis
- Cell communication analysis (CellPhoneDB)
- Gene Set Enrichment Analysis (GSEA)

Inner-sample analysis

Cell clustering:

Differential Expression Analysis: cluster DEGs

Inter-group analysis

Differential Expression Analysis: Group DEGs

Advanced Analysis:

Pseudotime analysis:

Cell cycle analysis: