The GSL offers RNA discovery and profiling services through next-generation sequencing and microarray analysis.
Upon arrival to the GSL, 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 GSL sample
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:
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).
Ribosomal Reduction RNA-seq: The rRNA reduction process removes structural 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).
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. During analysis with Tophat, GSL directional libraries would be analyzed as library type “fr-firststrand”.
Low input RNA-seq services: The GSL 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.
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. The GSL 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 GSL-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:
NextSeq, HiSeq 2500, and MiSeq by Illumina
Microarray experiments aim to characterize the expression of known mRNA starting with total RNA.
Current GSL Microarray Offerings:
BeadArray by Illumina
GeneChip PrimeView from Affymetrix