The SGT shared resource provides a large array of RNA sequencing services such Stranded mRNA-seq, smRNA-seq or ultra low input RNA-seq. If you do not find in the list below the RNA sequencing service you need for your project, please contact us.
Upon delivery to our facility, all RNA samples are evaluated for concentration by Qubit® and for integrity by 2100 Bioanalyzer, TapeStation or Fragment Analyzer. Before submitting samples, please review our sample submission requirements.
Choosing the correct RNA sequencing service for your samples is dependent on the organism, total RNA or number of cells available for input, and project goals. Contact us for questions regarding experimental design or RNA service options. Most next generation sequencing project requires special consideration. We do recommend you contact us before initiating a project with us.
- Stranded mRNA-Seq
- Total RNA-Seq with Ribosomal Reduction
- Small/mi RNA Analysis
- Ultra-Low-Input RNA-Seq
- Gene Expression of FFPE Samples
This is a popular tool for estimating gene expression levels and for comparing differential gene expression in model organism. mRNA-Seq can also provide valuable information about alternatively spliced variants and can contribute to identify novel isoforms. Among many other advantages, RNA-Seq allows for highly accurate expression quantitation within a wide dynamic range. We offer complete solutions for mRNA-Seq projects including experimental design, RNA-seq library preparation, and high throughput Illumina sequencing. We also offer analysis services and data storage in collaboration with the Genomic Analysis and Bioinformatics Shared Resource. We currently use the Kapa Stranded mRNA-seq library prep kit to make sequencing libraries and libraries are typically sequenced on Illumina instruments with a 50bp single end reads length. Paired-end sequencing with longer read length can also be done if one is interested in RNA-splicing.
For RNA extracted from humand blood, we are using the Nugen Universal Plus mRNA-seq kit with Globin AnyDeplete to prepare libraries.
Transcriptomes can also be assembled without the need of a reference genome. This approach is useful for determining gene sequences of non-model organisms. We offer services for performing whole transcriptome de novo sequencing on Illumina platforms and PacBio platforms. De novo transcriptome assembly using illumina platforms requires 150bp paired end sequencing. The long reads generated by PacBio sequencing provide full-length reads spanning entire transcript isoforms from the 5' end to the polyA-tail (see PacBio Iso-Seq description). The Iso-Seq approach does not require any assembly and provides with a full, accurate, and least biased transcriptome data.
Total RNA-Seq with Ribosomal Reduction
Total RNA-Seq with ribosomal reduction selectively removes ribosomal RNA from total RNA samples. In contrast to polyA+ enrichment mRNA-seq, this approach preserves non-polyadenylated RNAs allowing one to investigate broader classes of RNAs including immature mRNAs and non-polyadenylated ncRNAs. RNA-seq libarires are prepare using the Illumina TruSeq Stranded total RNA-seq kit in combination with a wide variety of ribosomal reduction kits (Illumina Ribozero kit) depending on the sample type:
- Ribozero Gold – removes cytoplasmic and mitochondrial rRNAs from human/mouse/rat samples
- Ribozero Globin – removes Globin mRNAs and cytoplasmic and mitochondrial rRNAs from human/mouse/rat
Small/mi RNA Analysis
Small/mi RNA analysis provides the ability to discover, measure and compare expression of know microRNAs and other small non-coding RNA. It also allows to detect novel microRNA targets. We use the Qiagen miRNA Library Prep kit for all the miRNA library prep.
Ultra-Low-Input RNA-Seq can be used to generate gene expression data from few or even single cells. Ultra-low-input RNA-Seq provides the opportunity to study differences between cells or cell types with an unprecedented resolution. This allows for a better understanding of biological differences between cells within a tissue/tumor and characterize subpopulation responses to environmental cues. We use the Clontech Ultra Low Input RNA SMARTer mRNA amplification Kit for the initial RNA amplification which provides a fast and simple method for preparing amplified cDNA from total RNA for RNA-Seq applications. The obtained dscDNA is then converted into a sequencing library using Kapa Hyper Prep Kit.
Gene Expression of FFPE Samples
Studying gene expression of FFPE samples is extremely valuable for better understanding diseases. Unfortunately, FFPE samples can be significantly degraded and traditional RNA-seq library preparation methods often fail to generate optimal libraries. We can use for your FFPE sample the Illumina RNA Exome kit, an alternative sample preparation method enabling the generation of high quality RNA-Seq results from degraded samples.