RNA Services
Discovery offers RNA discovery and profiling services through
next-generation sequencing and microarray analysis. Upon arrival to
Discovery, all total RNA samples are evaluated for
concentration by Qubit® or Ribogreen® and for integrity by 2100
Bioanalyzer or Caliper GX. Before submitting samples, please review
the Discovery sample submission requirements.
RNA sequencing
Choosing the correct RNA sequencing service for your samples is
dependent on the organism, total RNA available for input, and
project goals. For questions regarding experimental design or RNA
service options please contact us. If you plan to build a
transcriptome, please discuss your project with us before submission as
there are special considerations for such a project. We currently offer:
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Poly A RNA-seq: Standard Poly A RNA-seq libraries are prepared
for samples that require information from the expressed (transcribed)
RNA population. Poly dT bead selection is employed to specifically
target mRNA sequences, which limits sequencing data to Poly A
transcripts. The directional module may be used with this protocol
(see below).
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Ribosomal Reduction RNA-seq: The rRNA reduction process removes
tructural RNA from the total RNA population present in the sample.
rRNA reduction is employed to remove rRNA, which will allow sequencing
of the remaining RNA population. The directional module may be used
with this protocol (see below).
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Directional RNA-seq Directional sequencing requires the use of
Actinomycin D to inhibit DNA-dependent DNA synthesis so only RNA is
transcribed during first-strand synthesis. Additionally, dUTP is
substituted for dTTP in second strand synthesis to allow only the
first strand to be sequenced in the final library. This results in
stranded RNA-seq libraries, allowing assignment of a read to a
particular strand of DNA. The analysis of data from stranded or
directional libraries is straightforward and more precise than
non-directional libraries. All new projects are encouraged to add
the directional module to their RNA-seq studies as the cost is minimal
but the advantage is significant.
-
Low input RNA-seq services: Discovery uses the
Ovation RNA-Seq System V2 kit (Nugen) for initial RNA amplification
which provides a fast and simple method for preparing amplified cDNA
from total RNA for RNA-Seq applications. Our low input RNA-seq
service requires 1ng-10ng of total RNA with a RIN score of 7.0 or
higher. It allows amplification of both mRNA and non-polyadenylated
transcripts. After purification, the cDNA is fragmented to the
appropriate size and further library prep steps are followed to
prepare sequencing library. There is no directional module available
for this RNA-seq service.
- miRNA-seq library services: Our miRNA library prep service
requires 100-500 ng total RNA in 6ul volume with a RIN score of 7.0
or higher. We use NEBNext ®Multiplex Small RNA Library Prep Set for
Illumina® (NEB) coupled with automated agarose gel size selection
using the Pipin Prep Instrument (Sage Science) for the miRNA library
prep. This allows us to prepare individually barcoded miRNA libraries
with precisely size selected fragments free of adapter-dimers. There is
no directional module available for this RNA-seq service.
Sequencing Considerations
Poly A, ribosomal reduction, and low input RNA-seq are all usually
sequenced paired end, with standard read length of 50, 100, or 125bp.
Other sequencing conditions are available, but may require the purchase
of an entire Rapid Run or NextSeq run. In terms of coverage, for a
mammalian-sized genome, 25 million paired-end reads are usually
acceptable for reasonable coverage for poly A RNA-seq. Since ribosomal
reduction and low input RNA-seq include both mRNA and non-coding RNA,
more coverage is required to achieve a reasonable view of the
transcriptome. Discovery recommends at least 50 million paired-end reads
for these RNA-seq protocols. Small RNA-seq (microRNA-seq) is sequenced
at 50 bp read length (single end) to a depth of 15 million reads.
The ‘fragment size’ of a library refers to the portion of a library that is
located between the adapters and is sequenced. The poly A and ribosomal
reduction protocols include a fragmentation step using heat and chemicals,
which produces an insert size of approximately 185 – 215 bp. This fragment
size can easily be sequenced at 50bp, 75bp, 100bp, or 125 read lengths,
with the paired-end option if the analysis is amenable to some overlapping
reads. Low input or amplified RNA-seq samples are sonicated after
amplification to produce a fragment size of up to 400 bp. These libraries
would support up to a 125bp, paired-end sequencing run with little overlap
of reads. Fragment sizes for a library or set of libraries can be found in
two ways: 1. looking at the bioanalysis profile of the final libraries
under Results/Quality Analysis or 2. for a concise list, view the kapa
qPCR worksheet, which shows the average fragment size for all samples on
the first tab. The adapter length (combined) for Discovery-prepared libraries
is 119bp.
Libraries for a particular order may use all or part of a lane, with the acknowledgment that filling the remainder of a lane may require extra time in the sequencing queue. Current next-generation sequencing platforms for RNA-seq are:
NovaSeq 6000, NextSeq, and MiSeq by Illumina
Microarray Services
Microarray experiments aim to characterize the expression of known mRNA starting with total RNA.
Current Discovery Microarray Offerings:
BeadArray by Illumina
GeneChip PrimeView from Affymetrix
