Best practices for storage of bead normalized Nextera XT libraries

The Nextera XT protocol includes an optional bead-based normalization step for libraries after the PCR clean-up step in the protocol.

Because the bead-based normalized libraries are single-stranded after elution from the normalization beads, the libraries are not as stable as double-stranded DNA libraries, are more susceptible to degradation, and can adhere to the walls of tubes.

After bead-based normalization, libraries can be stored for up to 1 week at -25°C to -15°C.

To make sure that all library strands are completely denatured, perform a heat denaturation of the libraries immediately before sequencing, as follows, with the diluted library pool just prior to loading into the sequencing cartridge.

1. Incubate the denatured and diluted library at 96°C for 2 minutes (using a pre-heated heat block).

2. After the heat incubation, invert the tube 1-2 times to mix.

3. Quickly move the library to an ice water bath for 5 minutes. The quick cooling step helps to lock the sample in its single-stranded form.

4. Proceed immediately to cluster generation.

Note: For longer term storage of libraries, stop immediately after the PCR clean-up step when the libraries are still double-stranded, and store the SGP plate at -25°C to -15°C. Aliquot as needed to avoid multiple freeze-thaw cycles.

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