Total RNA / mRNA Sequencing
RNA-Seq is the premier tool for mapping and quantifying transcriptomes by utilizing next-generation sequencing (NGS) technology. The transcriptome refers to the complete set of transcripts in a cell, which provides information on the transcript level for a specific developmental stage or physiological condition. Understanding the transcriptome is necessary for interpreting the functional elements of the genome and understanding development and disease. The key purpose of transcriptomics includes cataloging all species of transcripts; determining the transcriptional structure of genes; and quantifying the expression levels of each transcript under different conditions.
RNA-Seq delivers an unbiased and unprecedented high-resolution view of the global transcriptional landscape, which allows an affordable and accurate approach for gene expression quantification and differential gene expression analysis between multiple groups of samples. RNA-Seq can identify novel and previously-unexpected transcripts without the need for a reference genome, allowing de novo assembly of new transcriptome that is not previously studied before. It also enables the discovery of novel gene structures, alternatively spliced isoforms, gene fusions, SNPs/InDel, and allele-specific expression (ASE).
Advantages of RNA-Seq
· Quantitative and precise measurements of RNA molecules at a single base-pair resolution
· Discovery of novel transcripts, splice variants, and gene fusions
· Application to any species, no matter whether reference genome is available
· A comparable or lower price than many other methods
Standard Analysis
· Statistics of sequencing depth and coverage
· De novo assembly, and reference genome mapping
· Gene annotations and gene expression levels
· Prediction of novel genes and identification of variants
· Differentially expressed genes (DEGs)
· A comparable or lower price than many other methods
Advanced Analysis
· Gene set enrichment analysis
· Alternative splicing
· Identification of fusion genes
Sample requirements
· Total RNA amount ≥ 1 μg; RIN: >6.5, OD260/280: 1.8-2.2; OD260/230: ≥ 1.8
Small RNA / miRNA Sequencing
Small RNA species generally include the most common and well-studied microRNA (miRNA), small interfering RNA (siRNA), and piwi-interacting RNA (piRNA), as well as other types of small RNA, such as small nucleolar RNA (snoRNA) and small nuclear RNA (snRNA). Small RNA is a type of lowly abundant, short in length (<200 nt), non-protein-coding RNAs that lack polyadenylation. Small RNA populations can vary significantly among different tissue types and species. Generally, small RNAs are formed by fragmentation of longer RNA sequences with the help of dedicated sets of enzymes and other proteins.
Small RNAs act in gene silencing and post-transcriptional regulation of gene expression. However, small RNA is not sufficient for the induction of RNA inference. It generally needs to form the core of the RNA-protein complex known as RNA-induced silencing complex (RISC). siRNAs can cleave the mRNA in the middle of the mRNA-siRNA duplex, and the resulting mRNA halves are degraded by other cellular enzymes. Unlike the siRNA pathway, miRNA-mediated degradation is initiated by enzymatic removal of the mRNA polyA tail. piRNAs are essential for the development of germ cells. Small RNAs have been demonstrated to be involved in a number of biological processes including development, cell proliferation and differentiation, and apoptosis.
Advantages of Small RNA Sequencing
· Small RNA and miRNA profiling
· Understanding how post-transcriptional regulation contributes to the phenotype
· Identifying more unmapped small RNAs and isoforms, as well as novel biomarkers
Standard Data Analysis
· Reference-based mapping
· Small RNA classification and quantification
· Expression profiling
· Differentially expressed miRNA
Advanced Data Analysis
· Target gene prediction and annotation
· Gene Set Enrichment Analysis
· Comparative data analysis
Sample requirements
· Total RNA ≥ 2 μg, OD260/280: 1.8-2.2; OD260/230: ≥ 1.8
· Exosomal RNA ≥ 100 ng, OD260/280: 1.8-2.2; OD260/230: ≥ 1.8
Single-Cell RNA Sequencing
Cells are the basic unit of life and is unique to each cell. The capacity in biological systems to expose complex cellular events is essential to a better understanding of cellular contributions during development or in progression of disease. Gene expression study on the single-cell resolution of samples made up of mixed cell populations provides a deep insight into the transcriptome nature of different types of cells.
Oneomics offers best-in-class methods for preparing and sequencing libraries from a single cell, a few cells and ultra-low RNA inputs. Combining robust cDNA synthesis technology with next-generation Illumina sequencing and analytics technologies, we offer high-quality, reliable data to research cell-to-cell transcriptome heterogeneity.
Bioinformatics Analysis
· Raw data quality control
· Statistics of sequencing depth and coverage
· Annotation and statistics
· Clustering
· Pathway enrichment analysis
Sample requirements
· 96-well plates of live cells suspension
· Frozen cells in lysates
· >200 pg extracted RNA, OD260/280: 1.8 to 2.2
PacBio Isoform Sequencing (Iso-Seq)
RNA sequencing (RNA-Seq) by short read is widely used for gene expression and isoform analysis. However, it is difficult to analyze full-length transcripts with short reads due to alternative splicing events and transcriptional regulation. With Pacbio’s SMRT sequencing technology, we can sequence the entire transcriptome from 5’ end to 3’ polyA-tail without assembly.
Advantages of Isoform Sequencing
· Discover novel transcripts & genes
· Identify fusion genes
· Resolve alternative polyadenylation
· Identify retained introns
· Find anti-sense transcription
· Annotate gene isoforms & alternative splicing events
· Recover missing exons
· Improve isoform-abundance quantification accuracy
Sample requirements
· Total RNA amount ≥ 5 μg (Concentration ≥ 300ng/μl), OD260/280: 1.8-2.2; OD260/230: ≥ 1.8