mRNA Library Preparation for RNA-seq


RNA-seq allows for quantiative transcriptomic profiling of biological samples. RNA-seq takes advantage of technological advancements in high-throughput sequencing by converting RNAs of interest to a library of cDNA fragments that can then be sequenced using standard DNA-sequencing technologies. Preparing a sample for RNA-seq typically begins with either rRNA depletion of mRNA enrichment. This is due to the fact that rRNA comprises a large percentage of total RNA and thus can take up valuable sequencing capacity making detection of the RNA species of interest more difficult.


mRNA enrichment
– polyA selection to pull down mRNA
– requires high quality, intact, non-degraded RNA or only the 3′ end of transcripts will be isolated
– requires large starting amount of total RNA (10 to 20 micrograms) since most of the total RNA will be rRNA

rRNA depletion
– depletes rRNA so non-coding RNA is preserved
– requires less starting amount of total RNA (1 to 10 micrograms)
– previous research has shown rRNA depletion to be “responsible for substantial, unappreciated biases in coverage introduced during library preparation”

Commercial protocols/kits

Illumina’s TruSeq RNA Sample Preparation kit v2
– uses polyT beads to isolate the mRNA from the rRNA, tRNA, and non-coding RNA
– use of these beads requires that the RNA be of very high quality or only the 3′ end of transcripts will be isolated
– purified mRNA is then fragmented with metal and random priming is used to convert the sample to cDNA

Epicentre’s ScriptSeq RNA-seq kit v2 with Ribo-Zero
– rRNA removal probes hybridize to rRNA
– magnetic beads bind with the rRNA removal probes to achieve rRNA-depletion

Commercial protocols/kits that do not use either mRNA enrichment or rRNA depletion

NuGen’s Ovation RNA-seq system v2
– utilizes non-random nonamers designed to not amplify ribosomal RNA to create double stranded cDNA fragments
– non-random nonamers cannot amplify all areas of the genome and certain portions of genes are often lost
– 2 to 200 nanogram of total RNA is needed

Clontech’s SMARTer Ultra Low RNA kit
– begins with cDNA generation using polyT priming and proprietary chemistry
– use of polyT priming requires the RNA to be of high quality
– full length double-stranded cDNAs are generated and amplified by PC
– very low starting amount of total RNA needed so ideal for single cell RNA-seq

So, what do you think ?