Sample Submission Form
Please fill out the Sample Submission Form and bring it when you drop off your sample.
Sample Requirements
If you would like sequencing core to prepare samples for sequencing, please see appropriate sample submission requirements below.
- DNA Sequencing: 100 ng or more for genome DNA, PCR products, BAC, YAC etc suspended in TE or EB Buffer in a volume not to exceed 50ul. DNA should be as intact as possible, with an OD260/280 ratio of 1.8-2.
- Targeted Exome Capture (Seq-cap): 1 – 3 µg of Purified DNA (5µg recommended) suspended in TE Buffer in a volume not to exceed 50µl. DNA should be as intact as possible, with an OD260/280 ratio of 1.8-2.
- Whole Transcriptome (mRNA-Seq) Profiling: 1 - 10µg of Purified RNA suspended in nuclease-free H2O in a volume less than, or equal to, 50µl. RNA should be as intact as possible, with OD260/280 ratio of 1.8-2 and an OD260/230 ratio of 1.8-2. Core lab will verify RNA integrity via an Agilent Technologies 2100 Bioanalyzer, regular RNA-seq, RIN ≥8.0; RIN≥9.5 for sample need to be treated with RiboMinus.
- Small RNA Querying: 1 - 10µg of Purified RNA suspended in nuclease-free H2O in 10ul volume. RNA should be isolated using a technique that preserves small RNAs (using Qiagen’s miRNeasy kit, for example). RNA should be as intact as possible, with OD260/280 ratio of 1.8-2 and an OD260/230 ratio of 1.8-2.
- ChIP-Enriched DNA Sequencing: 10ng of purified, ChIP-enriched, qPCR-verified, PicoGreen quantified DNA suspended in 30µl EB Buffer should be submitted.
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.
- TruSeq RNA Sample Prep Kit: 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.
Roche 454 kit protocol
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.
