SureSelect RNA Enrichment libraries are collections of oligonucleotides that can be used to enrich specific RNA species in a pool of RNAs for sequencing. You use a library of oligonucleotide "baits" that are complementary to the RNAs of interest (target RNAs). The target RNAs hybridize well with the baits, but other RNAs do not, which forms the basis of a powerful selection method that lets you focus your sequencing efforts. The enrichment protocol also preserves the relative abundance of targeted RNAs.
When you order a SureSelect RNA Enrichment library from Agilent, the final product that you receive is a kit that contains a set of biotinylated RNA oligonucleotides. However, as part of the library manufacturing process, Agilent first creates DNA oligonucleotides, and then later transcribes them into RNA. Thus, when you create a SureSelect RNA Enrichment library in eArray, you specify bait sequences in terms of DNA bases (A, C, G, T).
With eArray, you can access existing baits and bait libraries or create custom libraries of baits. The SureSelect RNA Enrichment application type in eArray supports the Applied Biosystems SOLiD platform and the Illumina sequencing platform. Multiplexing, which lets you sequence multiple samples in the same sequencing reaction, is also supported.
To use eArray to create bait libraries, you must be an eArray registered user. When you log in to eArray, you set the Application Type to SureSelect RNA Enrichment, and the relevant tabs and commands appear. When you become a registered user, you also gain access to eArray's large, robust set of microarray and library creation tools for a variety of different application types.
Note: You can also use eArray to create libraries that retrieve specific genomic DNA fragments for sequencing. See SureSelect Target Enrichment libraries.
In eArray, bait libraries contain one or more bait groups, and each bait group contains individual baits:
eArray stores individual baits as well as bait groups as separate entities, so you can use them as building blocks to make many different libraries.
For individual baits, you can use existing Agilent Catalog baits within eArray or your own baits. To put your own baits on eArray, you can upload them, and you can also use Bait Tiling, a method that creates baits that cover selected RNAs of interest, or the corresponding genomic regions, at even intervals. Currently, eArray supports a bait length of 120 nucleotides.
To organize baits into bait groups, you can select baits based on a Search Your Baits search. You can also create a bait group that contains baits that you upload or those that you create with the Bait Tiling tool. Baits and bait groups are separate entities—you can use a given individual bait in any number of bait groups.
One of the easiest ways to design bait libraries is to use one of eArray's built-in Library Wizards. Each wizard leads you step-by-step through the library creation process. As you complete a wizard, you can submit your newly-designed library to Agilent Manufacturing. To obtain the library, you can request a quote online through eArray. You can order the library from the Agilent Online Store or through your Agilent representative. eArray has several Library Wizards:
Create Library by Bait Tiling – Use this wizard to design a library that contains oligonucleotides that cover specific transcripts of interest, or the corresponding genomic regions, at even intervals.
Create Library from Existing Bait Groups – Use this wizard to design a library that includes existing collection(s) of baits (bait groups). You can include bait groups from your eArray account, and you can also include bait groups from the Agilent Catalog.
Create Library from Bait Upload – Use this wizard to create a library based on a file of bait sequences that you upload.
Combine Base and Supplemental Libraries – Creates a library that contains a selected base library and an additional library (a supplemental library). The base library can be one of several Agilent Catalog libraries, and the supplemental library can be any custom SureSelect RNA Enrichment library that is available to you in your eArray account. This lets you add baits for transcripts that are not represented in the catalog library.
eArray also gives you other options for libraries:
You can create a custom library manually from bait group search or browse results.
If your desired library already exists within eArray, either in the Agilent Catalog or in a folder to which you have access, you can search or browse for it. You can then submit it to Agilent Manufacturing and request a quote for it, or purchase it from the Agilent Online Store through eArray. Search and browse results also give you several other commands that you can use to manage libraries. See Actions you can take on libraries.
You can create a library to be used for multiplexing, which lets you sequence multiple input fragments in a single sequencing reaction. Multiplexing is available as an option for all custom SureSelect RNA Enrichment libraries. You select multiplexing when you request a quote for a library or purchase it from the Agilent Online Store through eArray.
eArray lets you publish SureSelect RNA Enrichment libraries on OpenGenomics.com, an Agilent-sponsored site that is dedicated to genomics research, news, and product information. The site contains a repository of microarrays and libraries that have been created by your colleagues around the world.
As you create libraries, they go through several status changes. When a library has a status of Complete or Submitted, eArray assigns it a unique Enrichment Library ID (ELID).
If you require greater flexibility, or want to focus your attention on one aspect of the process at a time, eArray lets you do this, as well. See the following topics for more details:
Baits
Bait groups
Libraries and library sets
Agilent provides these recommendations for the creation of custom SureSelect RNA Enrichment bait libraries:
The base coverage for the library should be from 0.2 to 3.2 Mb.
Use a tiling frequency of 2x.
eArray makes it easy for you to work with others down the hall, or around the world. You can create collaborations with others, then work together to construct and submit bait libraries of mutual interest. You create a special collaboration workspace that has many of the same tools as your individual user workspace. See these topics for more details:
Collaborations
What are the roles in a collaboration
Create a collaboration
The SureSelect RNA Enrichment application type is available in collaborations. However, the Bait Tiling tool within the SureSelect RNA Enrichment application type is only available in your individual user workspace.