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Single- nucleus RNA Sequencing

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 Spatial Transcriptomics chip and Illumina NovaSeq Platform

Service Details

Bioinformatics

Demo Results

Featured Publications

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:

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

Specifications

Sample Requirements

Library

Sequencing strategy

Data recommended

Quality Control

Cell number:

total > 10^⁶ or > 5x10⁵ flow sorted cells.

Tissue > 200 mg

Whole blood > 4 mL

10x Genomics sn cDNA library

Illumina PE150

100K PE reads per cell

(100-200 Gb)

700-1200 nuclei/μl and nuclei integrity observed under microscope

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

Service Work Flow

Previous: Plant/Animal Whole Genome Sequencing

Next: Genome-wide Association Analysis

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:

Explore the advancements facilitated by BMKGene’s single-nucleus RNA sequencing services by 10X Chromium in these featured publications:

Wang, L. et al. (2021) ‘Single-cell transcriptomic analysis reveals the immune landscape of lung in steroid-resistant asthma exacerbation’, Proceedings of the National Academy of Sciences of the United States of America, 118(2), p. e2005590118. doi: 10.1073/pnas.2005590118

Zheng, H. et al. (2022) ‘A Global Regulatory Network for Dysregulated Gene Expression and Abnormal Metabolic Signaling in Immune Cells in the Microenvironment of Graves’ Disease and Hashimoto’s Thyroiditis’, Frontiers in Immunology, 13, p. 879824. doi: 10.3389/FIMMU.2022.879824/BIBTEX.

Tian, H. et al. (2023) ‘Single-cell transcriptome uncovers heterogeneity and immune responses of leukocytes after vaccination with inactivated Edwardsiella tarda in flounder (Paralichthys olivaceus)’, Aquaculture, 566, p. 739238. doi: 10.1016/J.AQUACULTURE.2023.739238.

Yu, Y. et al. (2023) ‘Photodynamic therapy improves the outcome of immune checkpoint inhibitors via remodelling anti-tumour immunity in patients with gastric cancer’, Gastric Cancer, 26(5), pp. 798–813. doi: 10.1007/S10120-023-01409-X/METRICS.