RNA Sequencing

The Sequencing and Genomic Technologies shared resource provides a large array of RNA sequencing services, such as stranded mRNA-seq, smRNA-seq or ultra-low-input RNA-seq. If you do not find the RNA sequencing service you need for your project in the list below, 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 depends on the organism, total RNA or number of cells available for input, and project goals. Please contact us with questions regarding experimental design or RNA service options. Most next generation sequencing projects require special consideration. We recommend contacting us before initiating a project with us.

services


Stranded mrna-seq

Stranded mRNA-Seq is a popular tool for estimating gene expression levels and comparing differential gene expression in model organisms. mRNA-Seq  can also provide valuable information about alternatively spliced variants and can help identify novel isoforms. Among many other advantages, mRNA-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, which are typically sequenced on Illumina instruments with a 50 bp single end reads length.

Transcriptomes can also be assembled without a reference genome. This approach determines 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 150 bp 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 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 libraries are prepared 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 Bacteria (gram -) removes 23S, 16S and 5S rRNAs from Gram-Negative bacteria
  • Ribozero Bacteria (gram +) removes 23S, 16S and 5S rRNAs from Gram-Positive bacteria
  • Ribozero Yeast removes cytoplasmic and mitochondrial rRNAs from yeast
  • Ribozero Plant removes cytoplasmic, mitochondrial and chloroplast rRNAs from plants
  • Ribozero Epidemiology removes cytoplasmic and mitochondrial rRNAs from human/mouse/rat as well as 23S, 16S and 5S rRNAs from Gram-Negative and Gram-Positive bacteria
  • 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 expressions of known microRNAs and other small non-coding RNA. It also allows detection of novel microRNA targets. We use the Illumina TruSeq Small RNA Library Prep Kit coupled with agarose gel size selection for the miRNA library prep.


Ultra-low-input rna-seq

Ultra-low-input RNA-seq can generate expression data from few or even single cells. It provides the opportunity to study differences between cells or cell types with an unprecedented resolution, which allows for a better understanding of biological differences between cells within a tissue/tumor and characterizes 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 valuable for understanding diseases better. Unfortunately, FFPE samples can be significantly degraded, and traditional RNA-seq library preparation methods often fail to generate optimal libraries. For your FFPE sample, we can use the Illumina RNA Access kit, an alternative sample preparation method enabling the generation of high quality RNA-Seq results from degraded samples.


Related links:

Genomic Analysis and Bioinformatics Shared Resource