Does Tecan provide reagents for performing the bisulfite conversion step of the protocol?
The bisulfite conversion reagents are included with the purchase of the Ultralow Methyl-Seq System with TrueMethyl oxBS (Part No. 0541-32 and 9513-A01) and the Ovation RRBS Methyl-Seq System 1-16 with TrueMethyl oxBS (Part No. 0553-32). Other commercially available bisulfite conversion kits may be suitable as well, but these have not been validated.
Does Tecan provide reagents for performing the fragmentation step of the protocol?
Tecan does not provide the reagents used in the fragmentation steps. We suggest the Covaris instrument be utilized for DNA fragmentation, as suggested in the “materials” section of the User Guide.
Can I prepare MBBS1 and MBBS2 in advance/prepare extra solution?
It is recommended to prepare MBBS1 and MBBS2 fresh on the day of use. If reagent is prepared in advance or if excess reagent is prepared, store at 4 °C and use within one week. Discard after 1 week.
I don’t have access to a Covaris instrument. Can I use alternative fragmentation methods?
We have evaluated only Covaris fragmented DNA during the development of the Ultralow Methyl-Seq library prep kit. Other mechanical means of fragmentation, such as hydrodynamic shearing or nebulization, may be suitable.
Can I combine the barcoded libraries prior to the PCR amplification step?
This is not recommended. The stoichiometry of barcoded libraries may be adversely affected by this modification to the workflow. We suggest that the libraries be amplified and quantified independently before being pooled for use on the sequencer.
Why doesn’t my Library Amplification qPCR plot resemble the example in the user guide?
Certain real-time PCR instruments may display unexpected results, such as the example in the FAQ section of the User Guide. Ensure that your plot is set to display Rn. vs. Cycle, not deltaRn vs. Cycle, and that the y-axis is set to a log scale.