AMPure XP beads double-side purification

This step enriches for the nucleosome-free (~300 bp) as well as di and tri-nucleosome fragments. Removing small fragments (primer dimers) is important for optimal sequencing. First transfer 45 µL to an Eppendorf tube (or use PCR tube directly), add 22.5 μL (0.5X original volume, to remove large fragments) AMPure XP beads, pipet up and down 10 times to mix thoroughly. Incubate at room temperature for 10 minutes and place tubes in magnetic rack for 5 minutes. Transfer supernatant to new tube and add 58.5 μL (1.3X original volume, to remove small fragments) AMPure XP beads, pipet up and down 10 times to mix thoroughly. Incubate at room temperature for 10 minutes, place tubes in magnetic rack for 5 minutes and discard supernatant. Wash beads with 200 μL 80% ethanol (freshly made), pipet ethanol over beads 10 times, then discard ethanol. Ensure all ethanol is removed. Leave tube on magnetic rack with cap open for 3 to maximum 10 minutes depending on ambient humidity. The beads should be ‘glowing’ but not wet. Be careful not to over-dry them, which will decrease elution efficiency. Resuspend beads in 20 μL nuclease-free water, pipet up and down 10 times to mix thoroughly, place tube in magnetic rack for 1–5 minutes and transfer supernatant to new tube. This step can be replaced by Diagenode IP-Star, size selection 320 bp.

We have not systematically investigated if different purification procedures influence on the result. Purified libraries should be stored at -20°C and can be used for sequencing after up to 4 months.