Ampure xt beads

Library construction was performed according to the methods described by De Koker et al [17 (link)] with the following three modifications: [1] The complete eluate that was remaining after quality control (2 µL Qubit and 2 µL Femto PULSE) was used for library construction (= 71 µL). For this, samples were concentrated with a vacuum centrifuge (SpeedVac, Thermo Fischer Scientific, V-AQ programme) at 35°C and nuclease-free water was added to a volume of 11.1 μL. Unmethylated lambda phage DNA (0.005 ng, or 0.5 µL of a 0.01 ng/µL solution) was added to the eluate after the SpeedVac step. [2] Libraries prepared using the cf-RRBS protocol were cleaned by magnetic bead selection (AMPure XT beads – NEB) and eluted in 0.1× TE buffer. The libraries were visualized with the Fragment Analyser (Agilent) and quantified using the Kapa library quantification kit for Illumina platforms (Kapa Biosystems). [3] Based on the concentration, the libraries were equimolarly pooled and were sequenced on a NovaSeq 6000 instrument with a NovaSeq SP kit (paired-end, 2 × 50 cycles), using 3% phiX and a loading concentration between 1.8 and 2.5 nM. A maximum of 15 samples were pooled in one sequencing run, and samples from different donors and tubes were mixed to avoid sequencing batch effects.