Sample requirements for Illumina RNA-Seq libraries

Sample requirements for Illumina RNA-Seq libraries
rRNA Removal: The ScriptSeq v2 Kit produces directional sequencing reads using a random-primed cDNA synthesis
reaction. ScriptSeq RNA-Seq libraries can be made using polyA+ RNA or rRNA-depleted RNA from any animal, plant
or bacterial species. For rRNA depletion, we recommend using one of the Ribo-Zero™ rRNA Removal kits (Epicentre),
which are available for a wide range of species.
Unfortunately, for submission of already rRNA depleted or polyA enriched RNA samples, the Centre for Genomic
Research can take no responsibility for the level of data mapping to rRNA.
DNA Removal: DNA contamination of the RNA sample will greatly reduce the directionality of the sequenced library.
Therefore, we recommend treating the RNA sample with DNase I and remove the enzyme prior to the ScriptSeq
library prepation.
Accurate quantification of the nucleic acids in the sample(s) is necessary. Use a dye based method such as Qubit
(Invitrogen), or a gel based method such as the Agilent Bioanalyser.
Sample purity: Purification should be confirmed by values of ≥1.80 for both NanoDrop A260/230 and A260/280 ratios. If
the samples need further purification after submission, the additional expenses will be added to the formal quote.
Quality of the RNA: Please provide a gel image of all samples to confirm sample integrity. We recommend working
with intact total RNA with RIN values ≥ 7 as starting material for depletion or enrichment. This value is calculated by
the Agilent Bioanalyser software for most types of RNA. However, for some species the software cannot compute
the RIN value. In those cases, RNA integrity can be estimated by the sharpness of the rRNA bands and a baseline
value close to zero in the 200-1000 bp range. Whilst the RiboZero kit is reported to work with compromised RNA
samples (e.g. severely fragmented RNA from formalin-fixed, paraffin-embedded (FFPE) samples), we cannot
guarantee success with all FFPE RNA samples.
QC aliquot: Please supply a separate 2-µl aliquot for QC analyses to avoid defrosting the actual sample.
Required quantities of purified RNA in nuclease- and RNase-free water (free of DNA, contaminating salts, metal
ions, ethanol, and phenol):
Type of RNA
DNase treatment
Before submission
Total RNA
After submission
rRNA-depleted RNA
or polyA RNA
Before submission
After submission
Enrichment method
RiboZero kit
PolyA selection
RiboZero kit
PolyA selection
Not applicable
Not applicable
≥1 µg in 26-30 µl
≥2 µg in 26-30 µl
≥2 µg in 26-30 µl
≥4 µg in 26-30 µl
10-100 ng (ideally >50 ng) in 5-10 µl
20-200 ng (ideally >100 ng) in 5-10 µl