RNA-Sequencing enables rapid gene expression profiling of the transcriptome and allows for sensitive isoform-specific expression quantification.  RNA-Seq experiments are run on either Single Read (SR50) or Paired End (PE50) flowcells depending on the type of analysis you wish to perform.  For gene expression studies, samples are run on SR50 flowcells with 2-6 samples multiplexed per lane. For studies where isoform information is needed, samples are run on PE50 flowcells with 1-4 samples multiplexed per lane. The CCCB can perform RNA-Seq experiments on total RNA from cell lines or tissues, as well as on FFPE or other degraded samples. 

Sequencing Results

  • Raw Data:  Demultiplexed raw read in zipped fastq format
  • Quality Control Report: Sequence data quality assessment based on FastQC package, including per-base read quality, over-represented sequences, and over-represented k-mers
  • Alignment File: Sequenced reads are aligned to a reference transcriptome to produce an aligned BAM file. Because the alignments are made to the transcriptome rather than the genome, only known exons in known arrangements will be identified.  Basic processing will not identify novel transcribed regions or novel exon-exon junctions.   
  • Quantification:  Known genes and transcripts will be quantified in the sample using the Cufflinks algorithm, which produces FPKM values at the gene and transcript level.  All of the Cufflinks output will be returned to the user.

Standard Bioinformatics

  • RNASeq Quality Control: Assessment of the number of quantified genes, the distribution of gene quantification results, and overall sample clustering according to gene expression and quantification level
  • Differential Expression: Statistical analysis of differentially expressed gene and known transcripts with gene annotation
  • Gene Set Enrichment Analysis:  Determine if defined gene and pathways show statistically significant and concordant differences between two biological groups based on the broad GSEA algorithm
  • Ingenuity Pathway Analysis

Customized Bioinformatics

We can also perform customized analysis to meet specific needs of your projects.

Required Sample Inputs

NEBNext Poly A for Fresh Frozen Sample: 100ng total RNA
NuGen Ovation FFPE: 250ng total RNA
Clontech SMARTer Ultra Low: 250ng total RNA