CRC_br2
Sequencing libraries were prepared from 3 ng to 15 ng of cfDNA, depending on the clinical condition, using the NEXTFLEX Cell free DNA-Seq Kit (V2) (NOVA-5150-02) according to the manufacturers advice with one exception: the final library after PCR amplification was eluted in 20 L nuclease-free water. Library generation was performed with the Biomek FXP workstation (Beckman Coulter). Library quality was assessed by the Fragment Analyzer High Sensitivity DNA Kit (Agilent) and the concentration was measured by the Qubit dsDNA HS Assay Kit (Life Technologies). Size selection of cfDNA libraries was performed using a BluePippin instrument (Sage Science). To select the footprint DNA portion in the range of 150-200 bp, samples were applied to a 3% agarose BluePippin cassette according to the manufacturers protocol. Briefly described, 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 reamplified 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) (NOVA-5150-02), step C PCR amplification. Afterward, samples were purified with 1.2 x the volume of AMPureXP beads (Beckman Coulter) according to the manufacturers advice. Size selection performance was evaluated by the Fragment Analyzer High Sensitivity DNA Kit (Agilent). If the sample still contained fragments outside the target range of 150 bp to 200 bp, size selection was repeated as previously described, with the exception that the input sample was adjusted to 30 L with water and the reamplification step was performed with 5 to 8 cycles. After size selection, sequencing was performed on a NextSeq 2000 (Illumina) with 100 bp single end reagent kits.