742_11B
Total genomic DNA was isolated from HCT 116 clones using the Quick-DNA MIDIprep plus kit (Zymo Research) following the manufacturers instructions after cell collection by manual scraping in ice-cold PBS. Genomic DNA (250 ng) was fragmented in 50 ul microTUBES-50 (AFA fiber screw-cap) using an S220 sonicator (Covaris) following the manufacturers recommendation to generate 300-400 bp fragments. Fragmentation was followed by AMPure clean-up (0.9:1.0 beads:sample ratio) and 50 ng of DNA was used as input for library construction. Sequencing libraries were constructed using the NEBNext UltraTM II DNA Library Prep Kit (Illumina). Barcoding was performed using the indexing reagents provided in the NEBNext Unique Dual Index Oligos kit (Illumina). The final libraries were quantified by fluorescence (QUBIT, ThermoFisher Scientific), and sized on the Agilent TapeStation (DNA5000 Screen Tape). A yield of 50-60 ng of library was obtained of an average size ranging from 500-800 bp. Libraries were normalized, pooled equimolarly and treated with the Free Adapter Blocking Reagent (Illumina).