After SPRI bead purification of the crosslink-reversed samples, we transferred DNA from each to Covaris® microTUBE AFA Fiber Snap-Cap tubes (Covaris Cat. No. 520045) and sonicated to an average length of 400 ±85 bp using a Covaris® ME220 Focused-Ultrasonicator™. Temperature was held stably at 6°C and treatment lasted sixty-five seconds per sample with a peak power of fifty watts, ten percent duty factor, and two-hundred cycles per burst. The average fragment length and distribution of sheared DNA was determined by capillary electrophoresis using an Agilent® FragmentAnalyzer 5200 and HS NGS Fragment Kit (Agilent Cat. No. DNF-474–0500). We ran sheared DNA samples twice through the NEBNext® Ultra™ II DNA Library Prep Kit for Illumina® (Catalog No. E7645S) End Preparation and Adaptor Ligation steps with custom Y-adaptors to produce library preparation replicates. We purified ligation products via SPRI beads before Biotin enrichment using Dynabeads® MyOne™ Streptavidin C1 beads (ThermoFisher Catalog No. 65002).
We performed indexing PCR on streptavidin beads using KAPA HiFi HotStart ReadyMix (Catalog No. KK2602) and PCR products were isolated by SPRI bead purification. We quantified the libraries by Qubit™ 4 fluorometer and FragmentAnalyzer 5200 HS NGS Fragment Kit (Agilent Cat. No. DNF-474–0500) before pooling for sequencing on an Illumina HiSeq X at Fulgent Genetics.
We performed indexing PCR on streptavidin beads using KAPA HiFi HotStart ReadyMix (Catalog No. KK2602) and PCR products were isolated by SPRI bead purification. We quantified the libraries by Qubit™ 4 fluorometer and FragmentAnalyzer 5200 HS NGS Fragment Kit (Agilent Cat. No. DNF-474–0500) before pooling for sequencing on an Illumina HiSeq X at Fulgent Genetics.