RNA Sequencing
Assays from Tecan Genomics (previously NuGEN Technologies) and Illumina
The Single Cell Genomics Core Facility provides a RNA-Seq service, for investigators interested in using Next Generation Sequencing technology, for samples with small quantities of total RNA. For most projects our facility uses RNA –Seq protocols from Tecan Genomics and Illumina. Specifically the Tecan protocol, the Ovation RNA-Seq System v2, amplifies RNA samples between 500 picograms -10 nanograms, to create 2 -4 micrograms of double stranded cDNA. The Illumina protocol, the Nextera XT DNA Library Preparation Kit, is then used to create DNA library templates from the double stranded cDNA. The total cost per sample will include the cost for the amplification, library preparation, and sequencing. I would recommend using this service, if the total quantity of your RNA per sample is less than 300 nanograms, and/or if your lab is interested in sequencing both polyadenylated and non-polyadenylated transcripts. To use this assay the samples should have concentrations greater than 100 picograms / microliter. The minimum amount of RNA required is 500 picograms. However it is recommended to use at least 2 nanograms of total RNA per sample.
Assays from Takara (previously Clontech) and Illumina
Our Core Facility has also used the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara), for the initial amplification step for samples with low amounts of RNA (< 500 picograms of total RNA). This assay is designed to select and sequence polyadenylated transcripts. Specifically the Takara protocol, the SMART-Seq v4 Ultra Low Input RNA Kit, amplifies RNA samples with less than 500 picograms, to create 3 -17 nanograms of cDNA. The Illumina protocol, the Nextera XT DNA Library Preparation Kit, is then used to create DNA library templates from the cDNA. The total cost per sample will include the cost for the amplification, library preparation, and sequencing.
Sequencing Parameters
For either system there will be a RNA-Seq Processing Fee that includes the cost for the cDNA amplification, and library prep, per sample. There will also be a sequencing cost that will vary based upon the selected sequencing parameters (type of flow cell and selected read depth per sample).
The Single Cell Genomics Core reserves the right to wait and submit a pool that has the following number of samples:
- 15 -20M reads per sample: 12 samples per pool
- 25 - 30M reads per sample: 8 samples per pool
- 35 - 40M reads per sample: 6 samples per pool
If your project does not have the required number of samples for the pool, then your samples will be pooled with samples from another investigator.
