1. Services Overview
  2. Services
  3. Gene Expression and Transcriptomics
  4. Short Read RNA-seq Applications

Short Read RNA-seq Applications

The CGI offers multiple solutions for genome-scale transcriptomics, from RNA extraction through data generation and post-sequencing data analyses (through the Computational Biology Core). Sequencing is traditionally performed on Illumina short-read sequencing (SRS) platforms, depending on sample number and sequencing depth required. Please see INSERT LINK OUT for long-read sequencing RNA-seq applications. 

  • Total RNA extraction from cells of tissues
  • PAXGene total RNA – blood extraction 
  • High throughput RNA extractions (96-well format)
  • Assistance with Trizol extractions

Several different library preparation options are available in the CGI. If you are unsure which option is best suited for your sample(s)/experimental design, please contact us for guidance.  NOTE:   Library preparation kits include either polyA selection or ribosomal depletion so there is no need to enrich or deplete samples prior to submission unless your experiment requires it.

Library preparation kits validated and in use in the CGI include (but are not limited to):

  • Illumina Stranded mRNA Ligation
  • Illumina Stranded Total RNA Ligation
  • Takara Low Input SMART-Seq mRNA LP (generates unstranded mRNA libraries)
  • Takara SMART-Seq Total RNA Library Prep with ZapR Depletion (for Total RNA sequencing of highly degraded, LCM, FFPE or ultra-low input samples.
  • NEB Small RNA library preparation kit for miRNA sequencing

hort read sequencing is performed on the Illumina line of sequencing instruments available in the CGI.  The Illumina MiSeq or i100+ is utilized for library pool screening and/or library QC shallow sequencing for data analysis prior to deep sequencing submission. The NovaSeq X-plus is the preferred platform(*) for RNA-sequencing projects as it offers the most cost effective sequencing.   Flow cell choice (25B or 10B) is dependent on CGI sequencing queue.  If the CGI cannot fill a 25B flow cell, samples will move to a smaller flow cell (10B) so as not to delay data delivery.

(*) For projects requiring:

  • custom sequencing configurations (different that standard 150bp PE),
  • sequencing where lanes can not be shared due to incompatibility between library types, or
  • expedited turnaround time needed

… the CGI can provide pricing for NextSeq 2000 sequencing using X-LEAP chemistry. This platform offers a wide variety of sequencing kits starting at 50 cycles up to 600 cycles of sequencing on flow cells yielding as little as 200M total PE reads up to 3.6B total PE reads. 

Recommended sequencing depths for human-sized transcriptomes with good quality, DNA-free total RNA samples to start with are:

  • Stranded mRNA- 50M PE (**) reads
  • Stranded total RNA – 75M PE reads
  • Low input and/or degraded Total RNA – 75M PE reads
  • Low input mRNA (unstranded) 50M PE reads

(**) PE = Paired End reads.  For example, 50M PE reads would be a combined total of 25M read 1 reads + 25M read 2 reads.  The number of unique molecules being sequenced is 25M since read 1 and read 2 come from the same molecule.    

miRNA-seq (small RNA sequencing): 5-10M single end, 50bp reads are recommended for detecting known, highly-expressed miRNAs. 10-20M (or more, capping at 100M) single end, 50bp reads are recommended for discovery of rare or new miRNAs. Sequencing of miRNA-seq libraries is traditionally performed on a MiSeq, iSeq100+ or NextSeq 2000 sequencing platform using X-LEAP chemistry. 

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