RNA Services

RNA Services | Sequencing Data

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 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:

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. 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 Services

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