GSM2665658: iSLKr219_Dox_CaptureHiC; Homo sapiens; Hi-C
Chromatin prepared from KSHV-infected cells for the 3C procedure described above was used as input for Capture Hi-C. Briefly, infected TREx-K-Rta BCBL-1 or iSLK/r.219 cells (1 x 10^8) were fixed with formaldehyde, and the chromatin was digested with either BamHI or DpnII restriction enzyme and re-ligated under ultra-dilute conditions. DNA-protein crosslinks were reversed by incubation at 65°C overnight with 0.2% SDS. The DNA was then purified with the QIAquick PCR Purification Kit (Qiagen). Capture Hi-C sequencing libraries enriched for KSHV genome re-ligation products were prepared from DNA samples obtained from chromosome conformation capture (3C) 3analysis of TREx-K-Rta BCBL-1 and iSLK.r219 cells. The DNA preparations were sheared to a mean fragment length of 250 bp using a Covaris E220 Focused-ultrasonicator (Covaris, Inc.) and sequencing libraries were then prepared from the DNA chimerae using the NEBNext DNA Library Prep Kit (New England BioLabs, Inc.). Target enrichment for KSHV genomic content was performed using a custom-designed KSHV genomic capture probe library and according to the manufacturer’s standard protocols (IDT, Coralville, Iowa). Briefly, in-solution hybrid capture was performed by hybridizing libraries (500 ng) 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 at a temperature of 65°C for 4 hours. Hybridized targets were then bound to streptavidin-coupled magnetic beads (Dynabeads M-270 Streptavidin beads; Thermo Fisher Scientific) by incubation at 65°C for 45 minutes, and unbound DNA was removed by a series of high-stringency (65°C) and low-stringency (room temperature) washes. The KSHV genome-enriched Capture Hi-C library DNA was eluted and post-capture PCR enrichment performed with high-fidelity KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems, Inc., Wilmington, MA) and purified with Agencourt AMPure XP magnetic beads. KSHV-enriched libraries were then validated with an Agilent 2100 Bioanalyzer, and quantitated with a Qubit fluorometer (Invitrogen) and by qPCR-based quantification (KAPA Library Quantification Kit).
GEO
GSM2665658
GEO Accession
GSM2665658