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Blizard Institute - Faculty of Medicine and Dentistry

Transcriptome Analysis

Next Generation Sequencing - RNAseq

RNA sequencing is the method of choice for analysing global gene expression (we no longer run gene expression arrays). RNA samples are prepared into libraries using one of the methods outlined below. Once prepared these are sequenced on an Illumina Next Generation Sequencer to generate millions of reads per library which are then aligned to the genome.

When designing an RNA sequencing experiment, two important considerations are;
1) Which  library preparation method to use, this will affect the type of RNA captured and the number of reads per sample.
2) The number of sequencing reads per library.  A broad overview of gene expression can be achieved with 10 to 20 million read pairs per sample, increasing to 100 million read pairs per sample improves the power for isoform detection and to detect subtle transcription differences between samples.
If you would like to discus a project please email our centre manager Dr Charles Mein

Some of our publications in this area

  Library preparation type
Feature Total RNA mRNA Total RNA - Low Input mRNA - low input small RNA (inc miRNA)
Current Protocol NEB Next Ultra II NEB Next Ultra II   Clonetech SMART-Seq V4

Qiagen QIAseq miRNA

Priming Method Random hexamer priming Random hexamer priming   PolyT priming Ligation
Enrichment rRNA Depletion PolyA Selection      
Directional Stranded Stranded   Not stranded Not stranded
Degraded samples (eg from FFPE) Yes No Yes No Yes
Protein coding genes Yes Yes Yes Yes No
Long non-coding RNAs Yes No Yes No No
Small RNA (inc miRNA) No No No No Yes
Input amount 50-1,000ng 50-1,000ng 6pg-1ng 6pg-1ng 100-1,000ng
Recommended reads per sample 20-40 million 10-20 million 20-40 million 10-20 million 1-5 million
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