RNA-Seq is a next-generation sequencing (NGS)-based approach for profiling global gene expression patterns. It has many advantages over traditional methods, including the ability to produce highly accurate and quantitative readouts of gene expression levels for the entire transcriptome, sensitivity for even very lowly expressed genes, and the capability to identify alternatively spliced transcripts, novel transcripts and fusion gene transcripts. RNA-Seq is now widely adopted by researchers as the gold standard for transcriptome profiling in a variety of research contexts, including the analysis of genetically engineered animal models.
Cyagen offers transcriptome profiling by RNA-Seq as downstream services to your custom mouse/rat model generation. After you have made your animal models with Cyagen, we can breed your animals further to generate the desired genotypes, and perform RNA-Seq on various tissues of interest from these animals. Our services include tissue harvest, RNA isolation, RNA-Seq library preparation, Illumina NGS sequencing, and data analysis.
There are many benefits to Cyagen’s RNA-Seq services. With your custom mice/rats being bred in our facility, we can harvest tissues of interest freshly from your animals on-site and extract high-quality total RNA using optimized protocols, which is critical for obtaining good RNA-Seq data. Using our standardized RNA-Seq library preparation protocols and data analysis pipelines, we can deliver raw sequencing data, normalized gene expression data in a readable format, and results of differential expression (DE) analysis on different genotypes. By combining our highly affordable breeding services (which cost much less than most institutions) with our RNA-Seq services, you can obtain publication-quality RNA-Seq data at a fraction of the cost as doing it yourself. Importantly, you get your data with guaranteed success and fast turnaround.
- Knockout and knockin models: RNA-Seq are typically performed on three genotypes, including homozygous, heterozygous, and wildtype animals.
- Conditional knockout/knockin models: The animals need to be bred to deleter mice expressing tissue-specific Cre (or an inducible version of Cre such as CreER), and depending on the nature of the project, several genotypes could be used in RNA-Seq analysis.
- Transgenic models: RNA-Seq are typically performed on transgene positive animals and wildtype animals.
We can perform the necessary breeding and genotyping on your animals to obtain adequate numbers of animals for each genotype of interest. We recommend using three animals per genotype for RNA-Seq analysis to achieve adequate statistical power.
With your mice/rats housed in our animal facility, we can directly harvest fresh tissues of interest on-site and immediately isolate high quality total RNA. We will run Bioanalyzer RNA 6000 Nano chip to QC total RNA quality. For tissues on our standard tissues list (see below), we guarantee RNA integrity number (RIN) to be ≥ 7.0, which is qualified for conventional RNA-Seq library preparation. Non-standard tissues can be added upon request.
Standard tissues: brain, cerebellum, heart, liver, kidney, spleen, muscle, testis, ovary, blood, lung.
We use poly(A)-tailed mRNA isolated from total RNA to build RNA-Seq libraries. You can elect for us to construct either un-stranded or first-stranded RNA-Seq libraries. For un-stranded libraries, the resulting RNA-Seq data will carry no information regarding which DNA strand a transcript corresponds to. For most applications, RNA-Seq data obtained from un-stranded libraries are sufficient, because strand information is not used for obtaining the expression profiles of known genes. For first-stranded libraries, the resulting RNA-Seq data will reveal which DNA strand a transcript corresponds to. This is useful in some special applications, such as studying strand-specific transcription and detecting novel transcripts. By default, we perform QC by Qubit and Bioanalyzer on the final libraries.
Our standard sequencing format is 150 bp paired-end, and sequencing depth is ~20 million read pairs per library. Such sequencing depth is sufficient for conventional gene expression profiling and differential expression (DE) analysis. Additional sequencing depth can be requested. We will notify you by email when the sequencing is completed, and provide you with an FTP link for downloading raw sequencing data. We will keep your data on our FTP site for 3 months for free, but you can request to store your data for longer.
Having trouble dealing with raw RNA-Seq data? We can assist you with standard RNA-Seq data analyses, such as gene expression profiling and differential expression (DE) analysis. For gene expression profiling, we align read pairs to reference transcriptome, perform transcript assembly, and report normalized gene expression in FPKM (fragments per kilobase of transcript per million mapped reads) values. In addition, we can run DE analysis for your samples, and report differentially expressed genes between different genotypes with log fold changes and significance levels. Depending on your goals, customized data analyses such as identification of novel transcripts can be performed upon request. An FTP link will be provided to you for downloading data analysis results.
Our standard data analysis pipeline is shown below:
|Breeding animals to desired genotypes||Click here to view detailed description.|
|Tissue harvest||Tissues of interest are harvested and flash frozen in liquid nitrogen.||Free||2-4 days|
|Total RNA isolation and RNA-Seq library preparation||Total RNA isolation from tissues of interest, RNA-Seq library preparation, and related QC by Qubit and Bioanalyzer.||
$200 per un-stranded library
$240 per first-stranded library
|Illumina NGS sequencing||150 bp paired-end sequencing||$125 per 20 million reads (default)
$60 per 10 million additional reads
|RNA-Seq data analysis||Gene expression profiling and differential expression (DE) analysis||$120 per library for gene expression profiling
Additional $150 per DE analysis
* Turnaround time starts from when the animals are available in our facility (e.g. desired genotypes generated, desired age reached, etc.).
For RNA-Seq library preparation from our standard tissue list, we guarantee the input RNA RIN to be ≥ 7.0, and the final NGS library to be at ≥ 10 nM for 20 ul. For NGS sequencing, we guarantee the number of read pairs delivered to be ≥ 80% of the requested sequencing depth.
Please see below for supporting information related to Cyagen animal orders.