S32_T2
Short double-stranded cfDNA sequencing libraries were prepared from isolated cfDNA. In short, 15 ng of cfDNA were prepared using the NEXTFLEX Cell free DNA-Seq Kit (V2) (Perkin Elmer, Pittsburgh, USA) according to the manufacturers advice with the final library after PCR amplification was eluted in 20 L nuclease-free water. Library generation was automated using the Biomek FXP workstation (Beckman Coulter, Brea, USA). Library quality was assessed with the Fragment Analyzer High Sensitivity DNA Kit (Agilent, Santa Clara, USA) and concentration was measured by the Qubit dsDNA HS Assay Kit (Thermo Fisher, Waltham, USA). Size selection of cfDNA libraries was performed using a BluePippin instrument and the 3% agarose BluePippin cassette (Sage Science, Beverly, USA) according to the manufacturers protocol, thereby selecting footprint DNA in the range of 150-200 bp. Samples were filled up with water to 30 L and were mixed with 10 L of supplied internal marker (100 bp to 250 bp). 23 L of the eluted, size-selected sample was amplified with 25 L of NEXTFLEX PCR Master Mix 2.0 and 2 L of 1:10 diluted NEXTFLEX Primer Mix 2.0 as described in the NEXTFLEX Cell free DNA-Seq Kit (V2) (Perkin Elmer, Pittsburgh, USA). After purification of the size selected samples with 1.2 x the volume of AMPureXP beads (Beckman Coulter, Brea, USA) according to the manufacturers advice, a second size selection was performed as described above. Isolated, two times size selected samples were afterwards purified with 1.8 x the volume of AMPureXP beads (Beckman Coulter, Brea, USA). Size selection performance was evaluated by the Fragment Analyzer High Sensitivity DNA Kit (Agilent, Santa Clara, USA) and the concentration was measured by the Qubit dsDNA HS Assay Kit (Thermo Fisher, Waltham, USA). After size selection and molarity normalization of all samples, sequencing was performed on a NextSeq 2000 (Illumina, San Diego, USA) with 100 bp single read reagent kits.