RRC-CGF: Sample Submission
How to Place an Order for CGF Services
- Go to the UICore website and log in with your RRC (a.k.a. MyData) account name and password. If you do not already have a RRC account, you can find out how to register for one here.
- Click on the link for the Core Genomics Facility (CGF).
- Click on the service you would like from the list of services. If you are not sure which service to select, contact CGF for help.
- Click "Add to cart" and follow the on screen instructions to finish placing the order. For quantity, enter the number of samples (including replicates) that you are submitting.
How to Submit Samples to CGF
Samples can be delivered to CGF personnel during normal business hours after emailing the Sample Submission Form to us at email@example.com. Also include an excel table listing the sample names, 96-well plate addresses (when applicable), concentrations, volumes, OD 260, 280, 260/280, and 260/230. You should also to provide metadata for your samples (negative control, positive control, parent, etc.) if you will be relying on CGF for data analysis.
Please make sure tubes/plates are clearly labeled with accurate sample names. If there is a discrepancy between how the samples are labeled and the names listed in the excel table, we will use the names written on the samples.
After your service request is complete, take your remaining samples back. We will only store them for a month.
Service-Specific Sample Requirements
Fluidigm: Submitting Cells for capture on the C1 Single-Cell Auto Prep System
- Cells must be brought to us at the scheduled drop time. If cells are dropped off late, we may cancel the capture.
- Prior to scheduling a capture on the C1, we must QC your cells. During the cell QC we determine the cell size, viability, and buoyancy. We need to perform QC on each cell type or experimental condition because the treatment may alter the cell size and viability.
- Filter cells prior to submission with a 40 um cell strainer to remove clumps and debris.
- We need at least 500 ul of cell suspension at a concentration of 500-1000 cells per ul. Also, bring some of the cell culture media so we can dilute cells as needed.
- Before the capture, we will check the cell concentration and viability. Be available within 1 hour of the sample drop off in case cell viability is low and we need to make a decision on whether or not to proceed. If cell viability is significantly lower than recommended 80-90%, we may either cancel the capture or alternatively use 2 chips instead of 1 to capture a sufficient number of cells.
- For customers doing single-cell RT-PCR: Bring your pooled primers when submitting cells for C1 capture. Tell us how many primer pairs you have (you can have up to 93 assays, because three assays are needed for the RNA spike-in controls). Follow these instructions for pooling primers:
- Combine forward and reverse primers for each assay so that concentration of each primer is 100 uM. For example, mix 10 ul of 200 uM reverse primer with 10 ul of 200 uM forward primer. NOTE: You will need the 100 uM stock of F+R primers again when doing RT-PCR on the BioMark.
- Combine equal volumes of each primer pair that you prepared in the previous step. For example, for 93 assays combine 1 ul of each primer pair for a final volume of 93 ul.
Fluidigm: Submitting cDNA Samples for RT-PCR Analysis on the BioMark HD System
- Assays: Primers must be submitted in a 96-well plate. Forward and reverse primers should be mixed together in a single well at 100 uM each (so the total concentration of each well would be 200 uM). We need at least 5 ul of each primer pair per BioMark run. Leave 3 wells empty for the three RNA spike-in control assays.
- Samples: Preamplified cDNA samples must be submitted in a single 96-well plate. We need at least 5 ul of each sample per BioMark run. If your cDNA came from a C1 capture, then has already been preamplified. If you are using cDNA from other sources, then we recommend that you preamplify your cDNA unless you have in excess of 1,000 copies of your target template per ul of sample. Please contact us for a protocol for preamplification.
- Tube Controls: For each C1 capture we run a positive and negative "tube control", where the same primers and reagents that are used in the C1 chip for single-cell capture are then used to preamplify a few hundred cells in a single reaction using a standard thermal cycler. Theoretically, if you average the expression profiles from a hundred single cells, it should match the expression profile of the tube control. Let us know if you would like to include the tube controls in your BioMark run.
Affymetrix Arrays: Submitting Total RNA for Transcriptional or miRNA Profiling
- If you have very low concentrations of RNA (< 5 ng/ul), we can still profile your samples! We can profile as little 0.1 ng of total RNA on whole-transcript expression arrays. Contact us for more information about using samples with low concentrations.
- For RNA extracted from non-FFPE tissue, we need total RNA in water in the amount of 100 – 1000 ng with a concentration no less than 50 ng/ul, preferably 100 ng/ul or higher.
- For RNA extracted from FFPE tissue, we need at least 50 ng of total RNA in water with a concentration no less than 10 ng/ul, preferably 20 ng/ul or higher.
- RNA should not be degraded. High integrity eukaryotic total RNA run on a denaturing gel will have sharp 28S and 18S rRNA bands with the 28S band approximately twice as intense as the 18S band.
- RNA should be column or magnetic bead purified. If you would like us to do the column purification, consider that RNA loss could be as much as 70%.
- For miRNA profiling, please ensure that the columns used for RNA purification did not size select against smaller miRNA species. For example, regular columns provided with the Qiagen RNeasy Kit will not be appropriate for miRNA applications.
Affymetrix Arrays: Submitting DNA Samples for Cytogenetic Profiling:
- DNA must be pure, souble-stranded (verify with dsDNA-specific fluorescent dye such as PicoGreen), free of PCR inhibitors, and intact. We always verify DNA integrity on a gel and will consult with customers in case of problems.
- When extracting DNA, be sure to include an RNase treatment step.
- Submit at least 300ng DNA in no more than 6uL (≥50ng/uL).
Affymetrix Arrays: Submitting DNA Samples for Gene Regulation or Promoter Analysis
- Inquire lab personnel about specific submission guidelines for these platforms.
Agena (Formerly Sequenom): Submitting DNA Samples for Targeted Genotyping:
- DNA concentration should be 15-20 ng/ul.
- Total sample volume should be no less than 10 ul. For large projects (>30 SNPs), more volume may be required, please inquire.
- Samples should ideally be submitted in BioRad Hard-Shell HSP9631 (skirted) or HS9601 (semi-skirted) plates. If you would like to use different plates, please inquire with lab personnel.
Agena (Formerly Sequenom): Submitting DNA Samples for Methylation Profiling:
- Inquire lab personnel about specific submission guidelines for these platforms.
Illumina: Next-Generation RNA Sequencing
- Submit at least 5 ug total RNA, concentration should be at least 100 ng/ul. Best results are obtained when using RNA with an RNA integrity number (RIN) greater than 8.
Experion or Agilent Bioanalyzer: Submitting RNA Samples for QC
- Submit at least 2 ul for standard sensitivity analysis and 3 ul for high sensitivity analysis.
- For standard sensitivity analysis (quantitative range 25-500 ng/ul) sample concentration should be no less than 25 ng/ul.
- For high sensitivity analysis (quantitative range 100-5,000 pg/ul) sample concentration could be as low as 100 pg/ul.
Shipping Samples to CGF
- Make sure you have completed a Sample Submission Form. Email the form to us beforehand AND include a copy in the box with your samples.
- Pack the sample in a thermo-stable shipping box. A box with a wall thickness of at least 2 inches is recommended.
- RNA should be shipped frozen on dry ice and DNA should be shipped on ice packs.
- All shipping should be pre-paid by the user and shipments should only be made by FedEx or UPS.
- It is preferable to ship samples earlier in the week. Do not ship samples on a Friday.
- Ship to the following address:
- University of Illinois
- 835 S Wolcott Ave.
- E102 MSB MC 937
- Chicago, IL 60612
- ATTN: Zarema Arbieva/Room A302
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