Preparation of fragmented PhiX174 DNA
The sequencing libraries were prepared using PCR-free kits from 200 ng
of fragmented PhiX174 DNA. For the fragmentation, 1.5 µg of PhiX174 RF
II DNA (NEB, Ipswich, MA) was diluted in a final volume of 100 µL TE
10:1 in 0.5 mL Bioruptor microtubes and sonicated using Bioruptor
UCD-200 (Diagenode, Seraing, Belgium) at LOW power (160 W) during 12
pulses of 30s ON/90s OFF. A buffer exchange was performed with 10 mL
Tris-HCl pH8.0 using the kit NucleoSpin Gel and PCR Clean-up
(Macherey-Nagel, Düren, Germany). The profile of the fragmented PhiX DNA
was controlled on the Agilent Bioanalyzer 2100 instrument using the High
Sensitivity DNA kit (Agilent Technologies, Santa Clara, CA), and the
average fragment size was determined to be 289 bp. DNA was quantified
using the Qubit 1X dsDNA High Sensitivity Assay kit (ThermoFisher
Scientific, Waltham, MA) before library preparation.