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How to perform library heat denaturation (heat shock) before sequencing?
For amplicon-based libraries, highly structured or GC-rich libraries, and select other library types, cluster density consistency improves when the library is heat denatured before loading the pool onto the instrument. This method is optional for other libraries types as well.
Use the following steps to perform a heat denaturation. 1. After NaOH denaturation of the sequencing library, dilution with HT1 to the final loading concentration, and optional phiX addition, incubate the diluted library pool at 96Â°C for 2 minutes using a 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. This quick cooling step helps lock the library in its single stranded form. 4. Proceed immediately to cluster generation.