The Illumina Stranded Total RNA Prep is a commercially available kit from Illumina employed to construct RNA-Seq libraries from total RNA. Abundant rRNA (>90% of total) are enzymatically depleted before conversion of remaining RNA into cDNA. The protocol captures mRNA and non-poly A RNA such as long non-coding RNA (lncRNA). The approach can also be successfully used to sequence degraded samples and bacterial samples which lack polyA tails. Total RNA integrity is evaluated for each sample prior to library construction by processing on a Fragment Analyser to view the rRNA profile.
Ribosomal RNA depletion solutions are currently available for human/mouse/rat and bacterial samples. Contact the Genomics Core to discuss possible compatability of reagents with other species.
Unique dual-indexes are available for multiplexing of up to 384 samples. Total RNA input is 10 ng - 1000 ng.
Core staff are available to assist with experimental design and to answer questions related to next-generation sequencing technology. Arrangements for a meeting to discuss new or existing projects can be made by sending an email to Nathan Bivens, Genomics Core director.
All projects must be initiated with a quote before samples are submitted. Send a quote request to the general core email mugenomicscore@missouri.edu.
The RNA-Seq Calculator may be used to estimate the amount of sequence required for a project.
General Considerations:
- Submit samples only after paperwork has been completed.
- Clearly label tubes. Names must match those given on the project information form.
- Low binding microcentrifuge tubes (0.5 or 1.5 mL) are preferred for storing high quality nucleic acids.
Submission Requirements:
| Input Range | Submission Concentration |
Recommended Buffer |
Special Instructions |
| 25-1000 ng | 50 - 150 ng/μl total RNA | nuclease free water | Provide sample source and extraction methods used. DNase treatment strongly recommended. |
| Sequence Depth/Coverage* | Read Type | Index Type | Read Length | ||
| 50 million reads per sample | Paired end | unique dual-index | 100 bases | ||
* minimum read count recommended by MUGTC; increased sequencing depth may be desired for some studies