Sample Submission Form
Please fill out the Sample Submission Form and bring it when you drop off your sample.
Sample Submission
Prior to preparing any sample, we highly recommend you contact us and the Bioinformatics Core to discuss your project. A brief description of your project and detailed information about your samples and any QC data is required.
- DNA-seq: 100 ng or more with an OD 260/280 close to 1.8 for genome DNA, PCR products, BAC, YAC etc.
- ChIP-seq (chromatin immunoprecipitated material): 10 ng or more PicoGreen quantified material in 10 ml EB or TE buffer.
- RNA-seq: 100 ng double strand cDNA
- smRNA/miRNA: 1-10 mg total RNA or purified smRNA.
- Methylation: 10 ng enriched (e.g., MIRA enriched) DNA.
A high-quality library is critical to obtaining high quality data. Protocols used by the Core have been optimized. We also offer library preparation services.
The Core also designs 64 kinds of 6 bp- barcodes. Up to 63 samples labeled with different barcodes can be run in the same lane to decrease costs for those who need multiple samples with 30 million bases or less per sample.
Users interested in making their own libraries to expedite their studies should download and read the current protocols for different kits offered by Illumina (see below). Ordering information for kits is available upon request.
Illumina Kit Protocols
- Genomic DNA library: used to create sequencing libraries from genomic DNA, PCR product, BAC, YAC, and other plasmids.
- RNA library-NlaIII digested: used to create libraries from total RNA, in gene expression (transcriptional profiling) experiments.
- RNA library-DpnII digested: used to create libraries in gene expression (transcriptional profiling) experiments. Differs from previous protocols primarily in the use of one of the digesting enzymes. Both RNA library kits are very similar and simply reflect the historical use of each enzyme. Most users opt for this version.
- Small RNA library: used to create libraries to analyze low molecular weight RNA expression.
- ChIP-Seq library: used to create libraries for sequencing chromatin immunoprecipitation products.
Cluster Generation and Sequencing by Synthesis
Flow cell preparation and cluster generation on the cluster station is carried out by Core facility personnel. The amount of library DNA used at this step is the most important determinant for generating the maximal number of reads. Too few clusters will result in less sequence and too many clusters will cause signal interference during sequence imaging, resulting in excessive data loss. Achieving the optimal cluster density is difficult but critical to obtaining the greatest amount of high quality sequence data.
Based on our experience, qPCR is better than PicoGreen to determine the optimal concentration for your particular library to achieve the greatest amount of sequence. The sample may be run repeatedly through the cluster generation and sequencing steps until enough data is produced. Libraries will be kept at the Core facility in case additional data is needed from the same library.
