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