Submission Workflow Information
General Sample Submission Workflow
- Please read over this entire page first to get an idea about the process of preparing and submitting your sample(s).
- Contact Piotr Mieczkowski, Amy Perou, or Corbin Jones regarding your planned submission(s). They will be able to help you determine the appropriate technology for your project, offer suggestions on experimental designs, and get you started on the “right path”.
- Once you have determined the technology to be used for your project (Illumina, Ion, or PacBio), continue with the appropriate workflow links at the end of the General Sample Submission Workflow.
Recommended Kits for RNA and DNA Purification
The choice of purification method or kit for RNA or DNA purification can be critical to successful creation of your sequencing library. In our experience most commercial kits that use a column purification step can be used. Kits that rely on centrifugation of a resin out of the nucleic acid containing solution have a high failure rate since any trace carryover resin can inhibit downstream enzymology. For ethanol precipitation of RNA or DNA samples, never use glycogen as a carrier since this can unpredictably inhibit downstream enzymology and cannot be detected by typical assay methods. Instead we recommend using linear polyacrylamide (LPA) which is available from Sigma-Aldrich (Cat #56575) and Fisher Scientific (J67830-XF), as well as other vendors.
Kits that others have used with good success include:
Total RNA Isolation:
- PureLink™ RNA Mini Kit
- RNAqueous Micro
- MagMax™-96 Total RNA Isolation Kit
- Trizol® Reagent
RNA from FFPE:
- MagMax™ Total Nucleic Acid Isolation Kit
- RecoverAll™ Total Nucleic Acid Isolation Kit for FFPE
For Enrichment of RNA samples, we recommend:
For PolyA Selection:
- Dynabeads® mRNA DIRECT™ Micro Kit (not recommended with FFPE RNA)
For Ribosomal Depletion:
- RiboMinus™ Eukaryote System v2
- Low Input RiboMinus™ System v2
For Small RNA:
- PureLink™ miRNA Isolation Kit
- Qiagen Genomic‐tip kit (50-100 kb)
- Qiagen Gentra® Puregene® kit (100-200 kb)
- Phenol‐chloroform extraction
Ensure phenol is fresh and not oxidized; use within three months of opening the reagent bottle.
Sample quantification by NanoDrop or other spectrophotometer system has been found by us and others to routinely overestimate the amount of RNA or DNA present in samples. Our experience is this information can give a general idea of the sample concentration, it can over estimate by as much as 10-fold the true concentration of your sample. As a result, while we accept NanoDrop and other spectrophotometer readings as general guidance, all concentration values must be confirmed by a fluorometric assay, such as Qubit or qPCR for reliable accuracy. If you are unable to do this, the HTSF staff can do this for an additional charge. (Required concentrations are below.)
Note that a common reason for overestimation of concentration by NanoDrop (and other spectrophotometers) is failure to clean the pedestal or cuvette properly. See Technical Bulletin T031 from Thermo Scientific for proper cleaning and maintenance instructions.
Nucleic Acid Purity
It is essential that all submitted samples be of the highest purity and integrity. Testing for contaminants is performed by spectrophotometer by measuring A260/A280 and A260/A230 ratios. RNA in water should have A260/A280 >1.7 (2.0 is considered pure). DNA in water should have A260/A280 > 1.6 (1.8 is considered pure). Note that differences in solution acidity can result in fluctuations of +/- 0.3 for these readings. For both RNA and DNA A260/A230 should be >1.7 (expected 2.0-2.2). Lower values suggest the presence of contaminating phenols and/or carbohydrates. If you are unable to do this, the HTSF staff can do this for an additional charge. (Required purity is below.)
It is necessary to list the RIN (RNA Integrity Number) for most RNA samples.*
The RIN value is obtained from BioAnalyzer, TapeStation, Experion or Caliper LabChip assays. RIN value allows for classification of total RNA based on a numbering system from 1 to 10. The gradual degradation of rRNA is reflected by a continuous shift towards a broadening of the rRNA peaks and shorter fragments. The HTSF provides this service to researchers at UNC at our Carolina Crossing C location.
- Strongly Degraded = 3 RIN, DV200 > 50%*
- Partially degraded = 5 RIN
- Fully Intact = 10 RIN
- suggested min RIN Value = 8+
*FFPE RNA and other highly degraded samples have RIN values less than 3 or are even unreadable on a BioAnalyzer or similar systems. It is not always possible to predict which samples will be usable and which won’t from the RIN value due to the high degree of cross-linking and degradation. Illumina has developed a different metric (Illumina Evaluating RNA Quality white paper) for such highly degraded RNA samples, DV200 greater than 50% where DV200 is the % fragments greater than 200 nucleotides. This reading is obtained from your BioAnalyzer or TapeStation.
Required Sample Containers
Only 1.7 ml polypropylene snapcap tubes are accepted. We do not accept Matrix tubes, Dolphins tubes, or any tube ≥ 2.5 ml. If you have a question about whether or not we can accept a particular type tube not mentioned here, please contact the HTSF prior to preparing your samples.
The HTSF can ONLY accept BioRad (catalog HSP-9631) 96 well Skirted hard-shelled PCR plates. The HTSF can supply limited numbers of these 96 well plates as needed.
Sample Diluent Solution Requirements
Samples may be submitted in one of the following diluents:
- dH2O (preferred)
- EB buffer (10 mM Tris-Cl, pH 8.5)
- RSB buffer (10 mM Tris-Cl, pH 7.4, 10 mM NaCl, 3 mM MgCl2)
TE buffer is NOT acceptable and samples in this buffer will not be accepted or will incur an additional charge to correct this problem.
EDTA in these buffers interferes with downstream processing and can prevent the sequencing process from working correctly. Samples in TE buffer need to be cleaned up and re-suspended in an appropriate solution PRIOR to submission.
Samples should be physically dropped off at 1153 Genome Sciences Building on Mon-Fri between 10am and 4pm only please (unless otherwise arranged). Due to UNC security considerations, our facilities may not be accessible outside of these hours.