RNA analyses using Illumina Genome Analyzer

Sample Submission

Investigators are responsible for preparing sequencing libraries for digital gene expression-tag profiling and small RNA sequencing. The kit contains reagents for preparing cDNAs from RNA pools and preparing the sample for cluster generation. There are currently two protocols for digital expression analysis and one protocol for small RNA analysis. These protocols can be found below. Note: A protocol for preparing RNA for whole transcript sequencing is in development. Please contact the Genomics Resource for more details.

1) Digital Expression-Tag Sample Preparation Protocol:

• Using DpnII restriction digest
• Using NlaIII restriction digest

2) Small RNA Sample Preparation Protocol

For best results, researchers should closely follow Illumina's sample preparation protocols, including the specifications for final sample volume and concentration. Libraries should be submitted to the Genomics Resource (DE-740), along with the appropriate Illumina cluster generation and sequencing kits (see reagents section), and a completed submission form.

Note: Project timelines should be pre-arranged with the Genomics Resource prior to ordering reagents to ensure that your samples will be processed within your reagent's expiration window.

Genome Analyzer forms:
If you are using a FHCRC project id for billing, please fill out the Internal submission form

If billing is to be submitted to the University of Washington or other external institution, fill out the External submission form

Cluster Generation

Genomics Resource staff will introduce researcher's samples onto an Illumina Cluster Station to generate upwards of 40 million template clusters onto the flow-cell surface. The flow-cell is comprised of 8 independent lanes, of which one lane is dedicated to a phiX control sample supplied by the Genomics Resource.

Cluster generation is a critical step in the Illumina Genome Analyzer sequencing process. Too little sample introduced onto the cluster station will result in few clusters and therefore few template reads. On the other hand, too much sample will produce a high density of clusters that will be hard to distinguish from each other and will therefore dramatically reduce the number of reads. Unfortunately, the optimal concentration will vary from library to library and as such there are two approaches one can use to determine optimal sample concentrations.

Either:

• Use individual flow-cell lanes to perform a sample titration and assess the number of clusters generated. This approach is optimal and is well suited for large-scale studies requiring optimal sequencing performance. This approach will consume a cluster generation kit and a sequencing kit.

OR

• Use an estimated amount based on empirical data. This approach may not provide optimal results and in some cases may provide fewer reads than if the concentration were optimized for the sample under study.

Please contact the Genomics Resource for more information.

Continue in the process for RNA analysis using Illumina.