TOTAL RNA SEQUENCING

Sub-categories:

Total RNA-seq, transcriptome analysis, mRNA-seq, Long non-coding RNA-seq, RNA-seq for quantification & 3’end sequencing

Characterizing transcriptome dynamics by sequencing


While the genome in an organism is considered to be mostly static over time, changes associated with development, aging and disease is reflected in the transcriptome. Comparative studies applying RNA-seq enables investigation of changes in expression level, isoform usage and regulatory events as circularization.


While each project is unique and consequently the optimal solution varies accoring to the settings, some elements are will the be fairly consistent across most experiements. 

A complete omiics standard project for small RNA sequencing include:


Long RNAs

mRNA

lncRNA

circRNA

The project starts with a meeting with a NGS-scientist, where your research

and material is discussed and the optimal startegy is designed. 

  • RNA extraction 
  • Whole transcriptome enrichment by rRNA depletion*
  • Library preparation

              - 250-300 bp insert cDNA library
              - Multiple kit options available 

  • Stranded sequencing**

*   PolyA-enrichment or cDNA capture is also available, however, we highly recommend using rRNA removal as this provides data on both coding and noncoding RNAs. 

** Non-stranded sequencing is available, however, gives you less valuable data and while historical cheaper, the current prices are identical.


  • Platforms: HiSeq4000, HiSeq3000 or NextSeq500
  • ≥50 Million reads/sample (≥25 Million reads for 3’end-seq)
  • 100bp or 150bp PE sequencing (50bp/75bp SE for 3’end-seq)


PE: Paired-end, SE: Single-end

  • Quality control - scoring of reads and adaptor removal        (assessment of sequencing statistics)
  • Mapping to known genome/transcriptome (alignment) or De novo assembly (including sequencing assessment/alignment statistics)
  • Differential expression analysis and cluster analysis (Gene expression annotation, expression pattern analysis, Hierarchical clustering analysis, PCA analysis, quantitative expression profiles)


  • Tables (.excl*

  • Publication-grade figures (.TIFF*)

  • Written report summarizing the procedure and results done by omiics
  • Follow-up meeting with PhD-level NGS-scientist from omiics discussing results, suggestions for interpretation and input on possible next step(s) 

*   Other formats are also available if requested 


Additional

options

below

Additional Bioinformatic Analysis for Total RNA sequencing


  • Pathway analysis (Gene ontology analysis, Pathway enrichment analysis

  • Network analysis, including Condition specific expressed analysis, Gene Co-expression Network
    Analysis, Time series analysis

  • CircRNA detection and circRNA differential expression analysis

  • Alternative splicing/isoform usage analysis/Fusion gene analysis

  • LncRNA detection and function prediction /LncRNA Family prediction and classification

  • SNP and Indel detection

  • Protein-Protein Interaction (PPI) analysis

  • Comparison with online available data (Data-mining)