Automated DNA Library Preparation

RNA and DNA libraries were prepared from each of the selected samples according to the manufacturer’s instructions with the following exceptions. First, an enzymatic fragmentation was used for the preparation of DNA libraries instead of ultrasonication. The nucleic acid sample (up to 1 μg DNA, as measured by NanoPhotometer) was cut with the Nuclease P1 (New England BioLabs, Ipswich, MA, USA) as described previously [32 (link)], then cleaned with 1.8× AmPure magnetic beads. After the analysis of the fragment size distribution by the 5200 Fragment Analyzer System (Agilent Technologies, USA), if necessary, the sample was additionally cut for 5 min with the NEBNext dsDNA Fragmentase (New England BioLabs, Ipswich, MA, USA). The fragmented DNA (20–100 ng) was then subjected to the library preparation, starting from the end repair and A-tailing step. Second, instead of bead-based normalization, all prepared libraries were measured with the Qubit 4.0 fluorometer and the 5200 Fragment Analyzer System and pooled at equimolar concentrations for sequencing on the Illumina NextSeq 550 instrument.