Illumina Services

Guidelines for Sequencing Services

Samples for Library Preparation

Sequencing Libraries

RNA

The Genomics Core requires that total RNA be submitted for mRNA, small/miRNA RNA and total RNA sequence analysis. It is recommended that total RNA be purified using the TRIzol® Reagent with Phase Lock Gel^TM Heavy procedure (Trizol/Phase Lock Gel Protocol). If retention of miRNA is not required, the Qiagen RNeasy or other comparable isolation kits are acceptable. DNase treatment is required for RNA isolates used for Stranded Total RNA library preparation. DNase treatment of RNA samples intended for Stranded mRNA and Small RNA library preparation is strongly recommended to ensure accurate RNA quantitation. Submit RNA samples in nuclease-free H₂O at the following concentrations:

Stranded mRNA-seq: 100 - 250 ng/ul, 1-2ug Total RNA*
Stranded Total RNA-seq: 100 - 250 ng/ul, 1-2ug Total RNA*
small RNA-seq: 250 - 500 ng/ul, 2-3ug Total RNA*

*Note: Extra sample is appreciated for all RNA-seq library preparation procedures. The GSF requires that all RNA samples submitted for sequence analysis be assayed spectrophotometrically. All RNA samples submitted will be subjected to an Agilent TapeStation 4200 quality control run. Submit samples in 1.5 -1.7ml microcentrifuge tubes with the name clearly labeled on the top of the tube with permanent marker as it is written on the order form. Please limit sample names to eight characters. For large projects, submission in 96 well plate format must be accompanied with a plate map defining sample position.

gDNA / ChIP DNA

The Genomics Core recommends that genomic DNA (gDNA) submitted for sequence analysis be purified using the QIAamp DNA kit or comparable gDNA isolation kits. RNase treatment of gDNA is required. Submit gDNA samples in nuclease-free H₂O. Single ChIP enriched DNA or control input ChIP DNA should be submitted in nuclease-free H₂O. Submit either source DNA as follows:

gDNA-Seq - a concentration between 250 - 500 ng/ul, 1-2ug total*
ChIP-Seq - 30 ng Single ChIP enriched DNA / control input DNA in 30ul nuclease-free H₂O*

*Note: Extra sample is appreciated for all DNA-seq and ChIP-seq library preparation procedures.

Submit samples in 1.5 -1.7ml microcentrifuge tubes with the name clearly labeled on the top of the tube with permanent marker as it is written on the order form. Please limit sample names to eight characters. For large projects, submission in 96 well plate format must be accompanied with a plate map defining sample position.

Submission of Investigator Prepared Libraries

Please follow the listed conditions:

Libraries must be prepared using the TruSeq Library preparation kits or other kits compatible with Illumina SBS sequencing and clustering chemistry. For custom library constructs contact the Genomics Core before submission.
Libraries must be prepared with unique Dual Indexing. Both the i5 and i7 indexes must be unique within the individual library. The i5 and i7 indexes must also be unique from indexes used in other libraries. Prior to library preparation, index selection must be coordinated with the Genomics Core to avoid duplication with other projects. Failure to coordinate index selection may result in the delayed sequencing of your library submissions.
Accurate quantitation of library concentration is critical for flow cell clustering. It is mandatory that qPCR be used for library quantitation. The Genomics Core provides qPCR library quantification services. The Genomics Core will perform a mandatory qPCR library quantification to confirm the individual library or library pool concentration.
When multiplexing, individual library concentrations must be adjusted to 10 nM prior to pooling.
Investigator prepared libraries and library pools must be submitted at 10 nM concentration. Download the following conversion tool for converting ng/ul to nM: nM Conversion Calculator
It is recommended that an Agilent TapeStation or Agilent Bioanalyzer quality control run be performed on the library preparations to ensure proper sizing of library. Agilent TapeStation library validation services are available through the Genomics Core.
For individual libraries, please provide 20ul+ of 10 nM libraries in 1.5 -1.7ml microcentrifuge tubes with the name clearly labeled on the top of the tube with permanent marker as it is written on the order form. Please limit sample names to eight characters. For large projects, submission in 96 well plate format must be accompanied with a plate map defining sample position.

Sample Submission

Sequencing Libraries

Hand delivery of samples can be made between 9:00 am - 4:30 pm Monday - Friday at the Genomics Core located in RM# 1015 Hemenway, University of Kansas Medical Center. Complete the online NovaSeq 6000 order form prior to sample delivery. Dry ice shipments of samples, including completed order form, may be shipped overnight to the following address:

Genomics Core
1015 Hemenway
University of Kansas Medical Center
Receiving Dock
2106 Olathe Blvd.
Kansas City, Kansas 66160

For further information on the Genomics Core please browse the rest of our web site. Please feel free to call if you have any questions or require directions to the facility.

Genomics Core Contacts

Clark Bloomer, Project Manager - cbloomer@kumc.edu, (913) 588-7127
Rosanne Skinner, Sr. Research Associate - rskinner@kumc.edu, (913) 588-6927
Veronica Cloud, Ph.D., Research Associate – vcloud@kumc.edu , (913) 588-0711