How do I prepare DNA samples for RR-Seq (reduced representation sequencing)

Sample preparation:
If it has been established that enzyme (ApeKI) and method are suitable for the species your are working on (please see below), we require the samples for RR-Seq to be submitted in a 96-well plate.  One or two wells should remain empty for negative controls. The concentration of the samples should be normalized to 50 ng/ul as assayed by an intercalating dye (fluorometry using a Qubit; Quantus, or plate reader). The UV absorption ratios should be 260/280 nm 1.8 to 2.0 and 260/230 > 2.0 . A volume of 20 ul per sample is sufficient.    For GBS the DNA samples have to be extracted using a CTAB-free protocol (best a spin column protocol), since very precise DNA sample quantification is critical for the success of the protocol. The DNA samples have to be RNA-free. Thus, the DNA isolation protocol has to include an RNAse digestion step.
Before shipping us samples please email us gel images of representative samples. RR-Seq Sequencing is carried out with single-end 100 bp or single-end 90 bp reads (the latter cost the same as SR50 reads) on the Hiseq 4000.

Establishing the suitability of the enzymes and the protocol: 
At the moment we generate RR-Seq libraries with the ApeKI and CviAII enzymes only.   If there are no sequencing data for these enzymes for your species of interest yet, we would need to establish that the ApeKI or CviAII enzymes are suitable.  We will do this by carrying out test-library preparations (see below). However, before shipping us samples please carry out your own DNA restriction test in your lab and email us gel images of representative samples.  The gel images should show intact DNA samples as well as the same samples digested with a non-methylation sensitive restriction enzyme (e.g. HindIII).  Please see for example  https://www.neb.com/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions .   Agarose gel electrophoresis could be carried out on 1% gels with Ethidium bromide or GelRed staining. If the samples digest well, we would then do test library preparations experiments over here.  For these test please ship us two samples  (about 5 ug each at >50 ng/ul ; this test will cost $75).

 


Category: 03 Sample Preparation & Sample Requirements

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