Transcriptomics
RNA-Seq is a highly sensitive and accurate tool for investigating and discovering the whole transcriptome. It allows for differential gene expression profiling, identification of known and novel transcript isoforms, alternative splicing events, gene fusions, and post-transcriptional modifications. We offer services for total RNA-Seq, mRNA-Seq, and small RNA-Seq, and for high and low quality samples. All standard services include RNA fluorometric quantification and integrity analysis, library preparation, library fluorometric quantification and fragment analysis, and Illumina single-end sequencing. For non-standard services, please contact genomics@cshs.org. We also offer single cell RNA-Seq options.
Total RNA-Seq
Total RNA-Seq measures the full complement of transcripts in a cell, including mRNA and non-coding RNAs, for a comprehensive view of the transcriptome. Researchers are able to determine gene expression levels of each transcript, identify introns, exons and their boundaries, splice variants, and capture novel features of the transcriptome. Ribosomal RNA is removed prior to or during library construction in order to focus on valuable RNA species of interest. In the case of RNA from whole blood, globin RNA is also removed. cDNA synthesis is performed using random primers. Sample requirements and read outputs can be found here.
mRNA-Seq
mRNA-Seq enriches for polyadenylated (poly-A) transcripts of the transcriptome, including mRNAs and long non-coding RNAs with a poly(A) tail, via purification and cDNA synthesis with poly(dT) oligomers. This enrichment increases sequencing depth by focusing only on coding genes, improving the sensitivity towards identifying rare variants and lowly expressed mRNA transcripts. Our mRNA-Seq service is appropriate for high quality RNA samples. For high quality RNA in low amounts, we offer a low-input mRNA-Seq option, which also allows for intact cells as the input material (see low-input mRNA-Seq). For degraded RNA, such as from FFPE sections, we offer a 3’ end counting with unique molecular index (UMI) strategy for gene expression studies (see 3’ mRNA-Seq for degraded or FFPE RNA). Sample requirements and read outputs can be found here.
Low-input mRNA-Seq
For samples in limited quantity, we offer low-input mRNA-Seq. Our workflow generates high-quality cDNA from ultra-low amounts of total RNA, or directly from 10-1,000 intact cells. It offers unparalleled sensitivity and unbiased amplification of cDNA transcripts, enriches for full-length transcripts and maintains the true representation of mRNA transcripts from the original sample. The same workflow is available for single-cell mRNA-Seq of the full transcript. Sample requirements and read outputs can be found here.
3' mRNA-Seq for Degraded or FFPE RNA
For moderately to severely degraded RNA, such as that from FFPE samples, we offer a robust solution for mRNA-Seq from degraded RNA. Our workflow features a high correlation in genes detected between matched fresh frozen and FFPE samples, low input option, unique molecular index (UMI) for unbiased and accurate quantification, low read requirement, and high multiplexing options. This option is suitable for gene expression studies only. Sample requirements and read outputs can be found here.
miRNA-Seq
miRNAs (15-30 nt) act in gene silencing and post-transcriptional regulation of gene expression. qPCR and microarrays are traditional techniques used to asses miRNA expression. Unlike qPCR, however, miRNA-Seq does not require a priori knowledge of the miRNA sequence, allowing for novel discovery. Mature miRNAs contain a 5’phosphate and 3’hydroxyl group that is utilized to selectively target and enrich miRNA species. Incorporation of a unique molecular index (UMI) enables unbiased and accurate quantification of mature miRNAs. Sample requirements and read outputs can be found here.