The UM DNA Sequencing Core can convert your RNA samples to cDNA pools. By sequencing those pools, you can then count the number of times each species of cDNA in the pool is “tagged”. That enumeration reflects the expression levels of the RNAs present in your original sample. The sequences detected are not dependent on prior expectations, so you can detect novel and previously-unexpected RNA species. The potential dynamic range of the counting data is limited only by the total number of sequencing “tags” collected, so with enough money you can detect RNAs that are present at fewer than one copy per cell. With proper experimental design, you can even make inferences about allele-specific expression or alternative splicing events.
There are many variations on the basic concept of RNA-seq. The library preparation might be polyA-based, or might instead use ribo-reduction methods to see non-adenylated RNAs. It may generate strand-specific libraries, or it may yield nonstranded libraries. Costs per sample can be minimized by multiplexing several samples into each sequencer lane, using “barcoded” oligo adapters during the library step to allow for subsequent parsing of sequences from each sample. Alternatively, extremely deep sequencing can be selected, in order to allow detection of unusual or rare RNA events. Your Bioinformaticist should be consulted in order to choose from among the many possible library and sequencing options.
RNA_seq requires (1) one library preparation for each sample, then (2) typically requires multiple such libraries to be mixed (multiplexed) together, after which (3) the mixture is sequenced. Each of those three steps incurs a cost, and each requires you to make choices, summarized below.
Library Options: Consult with your Bioinformaticist to select one of these options:
Basic, non-stranded mRNA-seq: You provide a sample of total RNA, and we will create and sequence a library from the polyadenylated mRNAs present.
Strand-specific RNA-seq: You provide a sample of total RNA, and we will make the library in a way that allows you to later determine which genomic DNA strand was transcribed into the RNA. We can do this using either polyA selection, or ribo-depletion.
Ultra-low non-stranded RNA-seq: Similar to ‘Basic’ RNA-seq (above), but we can start with a very small amount of RNA, yielding non-strand-specific sequence information.
Ultra-low strand-specific RNA-seq: Similar to the ‘Strand-specific RNA-seq’ described above, but we can start this method with a very small amount of RNA, preserving strand-specific information.
smRNA-seq: You provide a sample of total RNA, and we will create and sequence a library so that you will see primarily the small RNAs present in your sample (e.g. siRNA and similar).
IsoSeq: If we use a long-read sequencer (the Pacific Biosciences RS II) we can obtain the sequence of very long segments of mRNAs. From these, you can determine which splice isoforms are present in your sample.
Sequencing Options: Consult with your Bioinformaticist to select one of these options:
Single-end, short-read sequencing: This is the most common choice for sequencing RNA-seq samples. The cost is comparatively low, but the number of sequences generated is excellent. In many cases, you don’t need the more expensive paired-end or long-read sequencing.
Paired-end, long-read sequencing: This is the second most common choice for sequencing RNA-seq samples. The cost is comparatively high, but with longer sequence reads and paired ends, you will have better distinction between of closely-related gene family members, improved detection of allele-specific expression, and improved detection of alternative splicing events.
Other sequencing options: We can also do many other types of sequencing, for example paired-end short-read sequencing, or very long read sequencing. These options generally carry penalties in terms of price, data yield and/or turnaround time, but may be important for specific studies. Contact the Core Director for more information