GSM4984016: BCBL-1_latent_CHi-C_Rep2; 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 then sheared to an average length of 400 bp using a Covaris E220 Focused-ultrasonicator (Covaris, Inc.) and fragments size-selected with AMPure XP beads to achieve a size distribution of 200-600 bp. The biotin-marked ligation products were then enriched by affinity capture with streptavidin magnetic beads (DynaBeads MyOne Streptavidin C1; Invitrogen, Thermo Fisher Scientific). Subsequently, libraries were prepared from the bound ligation products with the Kapa HyperPrep Kit with Library Amplification Module (Roche) using an on-bead modification to the standard protocol for end repair, dA-tailing, and ligation of Illumina TruSeq sequencing adaptors. The KSHV CHi-C library was then prepared by target enrichment of the libraries for KSHV genomic content by solution hybridization with a custom-designed KSHV genomic capture probe library (xGen Lockdown Probes; Integrated DNA Technologies, Inc., Coralville, IA) as previously described (13). Briefly, libraries (500 ng) were hybridized with the KSHV genomic capture probe pool (3 pmol) in a mixture containing xGen 1X Hybridization Buffer, Cot-1 (5 µg), and xGen Universal Blocking Oligos for 4 hours at 65°C. The hybridized targets were then captured with streptavidin beads (DynaBeads MyOne Streptavidin C1; Thermo Fisher Scientific) by incubation for 45 minutes at 65°C. Unbound DNA was removed by a series of high-stringency (65°C) and low-stringency (room temperature) washes. 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, Inc., Wilmington, MA).