RNA library preps

RNA preps we offer:

• Watchmaker mRNA library prep (96 samples) (Cat. nr 7BK0001-096), Watchmaker + adapters: xGen™ UDI Primer Pairs - xGen™ UDI 10nt Primer Plates 1-4 (Cat. nr 10008052), IDT
• TruSeq® Stranded mRNA Library Prep (96 Samples) (Cat.nr. 20020595), Illumina + adapters: IDT for Illumina – TruSeq RNA UD Indexes (96 Indexes) (Cat.nr. 2002237), Illumina
• QIAseq miRNA Library Kit (96) (Cat.nr. 331505) + adapters: QIAseq miRNA NGS 96 Index IL (96)- (Cat.nr. 331565)

Input requirements for RNA library preps.

1. mRNA-seq: Users should submit total RNA. We perform the standard Watchmaker mRNA prep (we are currently phasing out the TruSeq Stranded mRNA preparation kit, which Illumina has announced will be discontinued in 2027)as a service, which employs poly-T beads to enrich the polyadenylated fraction of mRNA. If the use of poly-T beads is not appropriate for your sample, it is your responsibility to inform us of this. 

2. Total RNA-seq: For samples where poly-T selection of RNA is inappropriate (eg pre-isolated mRNA / viral samples / rRNA-depleted samples). You supply your RNA, and we enter the RNA-seq prep at the appropriate stage in the protocol, omitting the poly-T bead step. It is critical that the RNA is in this case supplied only in water. 

Note that we cannot guarantee successful prep of rRNA-depleted samples, as it is usually impossible to distinguish successfully depleted samples from degraded RNA. These library preps are therefore performed at the user's risk.

3. small RNA-seq: Total RNA must be prepared by a method that retains small RNAs. Various vendors provide kits and protocols with adaptations to retain small RNAs. Avoid precipitating RNA, as this reduces the content of small RNAs.

Due to large variations in miRNA amounts in different tissues, success of miRNA preps is not guaranteed. Users submitting total RNA will therefore be billed for reactions consumed even in the event we are unable to prepare libraries. In the event that preps from total RNA are unsuccessful, users may attempt to enrich the small RNA fraction by PAGE or column fractionation and submit this instead, but be sure to note this in the submission form

rRNA depletion: We also offer rRNA depletion using Qiagen’s FastSelect reagents (QIAseq FastSelect ) on total RNA you supply. For human/mouse/rat RNA, we can offer this by the reaction and keep reagents in stock. Other depletion reagents (16S/Globin/Fly/Worm/Yeast/Fish/Plant) will most likely need to be ordered specially for your project and will incur at least the cost of the smallest kit size (8-24 reactions). It is critical that the RNA is supplied in water, minimum 200 ng in 10 μl. 

Sample amounts 

As a general rule, more RNA is better, so please provide more than the recommended sample amounts listed below if you can. Using the minimum amount increases the chances of failed preps. We may also be willing to attempt library preparation with amounts lower than the given minima, but this must be agreed with us in advance of submission, and you must nonetheless pay the cost of consumed reagents in the event of prep failure.

Sample integrity

Evidence of sample quality is mandatory before submission, unless agreed in advance. A BioAnalyzer trace/list of RIN numbers is preferred. Agilent Bioanalyzer RNA Integrity Number (RIN) should be > 7. Gel images showing rRNA are also acceptable, in which case samples should have a 28S/18S ratio >1.6.

Sample purity

For DNA and RNA samples the 260/280 ratio should fall in the range 1.8-2.1 and the 260/230 ratio within 1.8-2.4. We strongly recommend that RNA be prepared by procedures that include a DNase digestion to remove contaminating DNA.

Accepted buffers

See table. We do not accept lyophilized or precipitated samples. Samples delivered in inappropriate buffers will be returned to the user.