GSM4984004: BCBL-1_TREx-K-Rta_latent_CHi-C_Rep1; Homo sapiens; Human gammaherpesvirus 8; Hi-C
Chimeric DNA ligation products were prepared for KSHV Capture Hi-C (CHi-C) using kitted reagents from Arima Genomics (San Diego, CA) based on methods described for in situ Hi-C and CHi-C. Briefly, cells were crosslinked with 2% formaldehyde, lysed, and the genomic DNA digested with a cocktail of 4-cutter restriction endonucleases by incubation for 30 minutes at 37°C. The 5'-overhangs were then filled in and labeled with biotinylated dATP (biotin-14-dATP) by incorporation with Klenow fragment of DNA polymerase I (incubation for 45 minutes at 25°C). Ligation of the spatially proximal blunt-ended fragments was then performed with T4 DNA ligase (incubation for 15 minutes at 25°C). The formaldehyde crosslinks were reversed and the proximally-ligated, chimeric DNA products were purified with Agencourt AMPure XP paramagnetic beads (Beckman Coulter). Hi-C and KSHV Capture Hi-C (CHi-C) libraries were prepared from the chimeric DNA ligation products using kitted reagents from Arima Genomics (San Diego, CA). The DNA was fragmented to an average size of 400 bp with a Covaris E220 Focused-ultrasonicator (Covaris, Inc.) and size-selected to have a fragment size distribution of 200-600 bp with AMPure XP beads. The biotin-labeled ligation products were then selectively enriched by affinity capture with streptavidin magnetic beads (Arima Enrichment Beads). Subsequently, sequencing libraries were prepared with the Kapa HyperPrep Kit with Library Amplification Module (Roche) using a modified protocol for on-bead end repair, dA-tailing, and ligation of Illumina TruSeq sequencing adaptors. The KSHV CHi-C library was then prepared from the above Hi-C libraries. Briefly, target enrichment for KSHV genomic content was performed by solution hybridization with a custom-designed KSHV genomic capture probe library (xGen Lockdown Probes; IDT) and subsequent capture of the hybridized targets with streptavidin beads (DynaBeads MyOne Streptavidin C1; Thermo Fisher Scientific) according to the manufacturer's standard protocol (IDT). The KSHV genome-enriched CHi-C library DNA was eluted and PCR enrichment (12 cycles) performed with high-fidelity KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems).