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| Status |
Public on Mar 17, 2022 |
| Title |
iSLK_ORF57-KO_lytic_CHi-C |
| Sample type |
SRA |
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| Source name |
ORF57-KO iSLK
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| Organisms |
Homo sapiens; Human gammaherpesvirus 8 |
| Characteristics |
tissue: human epithelial cells
cell line: iSLK epithelial cells
cell type: Stable doxycycline-inducible K-Rta expression and infected with ORF57-KO KSHV BAC16
viral infection: KSHV
virus status: lytic
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| Growth protocol |
ORF57-Wt iSLK and ORF57-KO iSLK cells were cultured in complete DMEM supplemented with 10% FBS. BC3 and BC1 cells were grown in RPMI 1640 medium supplemented with 15% FBS. TREx-F3H3-K-Rta BCBL-1 (TREx-K-Rta BCBL-1, which have tetracycline/doxycycline (Dox)-inducible expression of N-terminal Flagx3- and HAx3-tagged K-Rta, were cultured in complete RPMI 1640 (supplemented with 15% FBS) containing blasticidin (10 μg/ml) and hygromycin B (200 μg/ml). TREx-F3H3-K-Rta BCBL-1, ORF57-Wt iSLK and ORF57-KO iSLK cells were treated with or without 1 µg/mL doxycycline for 28 h.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
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).
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| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Description |
Homo sapiens; KSHV
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| Data processing |
Basecalling and quality scoring were performed using Real-Time Analysis software (RTA2).
The HiC-Pro v2.11.1 pipeline was used to process the CHi-C data by performing read alignment, detection and filtering of valid interaction products, binning, and contact map normalization. Read alignment was performed with bowtie2 using a combined assembly of the human hg19 (GRCh37) and KSHV (NC_009333.1) reference genomes and by first mapping each read independently. The reads were filtered to remove duplicates and to keep only reads that mapped to the KSHV genome by using the Python module Pysam v0.14.1. Valid Hi-C interactions were detected based on several criteria, including the assignment of each read to a different restriction fragment and being located within a specified distance to a restriction site consistent with the size distribution of the sheared ligation products. Read pairs from invalid ligation products (e.g., self-circle ligation, dangling end, random breaks) and PCR duplicates were discarded. The valid reads were stored as matrices and binned with resolution of 500 bp (2 kb for density plots), the bins with more than 100 counts and at least 75% of cells with no-zero counts were used in the next steps. Iterative Correction and Eigenvector decomposition (ICE) normalization was used to treat the data with iteration of 100.
Genome_build: GRCh37 (hg19) + KSHV genome (GQ994935.1)
Supplementary_files_format_and_content: Processed data files are in hic format, which is an indexed binary format that is generated from Hi-C tag directories. This file format is designed to permit fast random access to contact matrix heatmaps in Juicebox.
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| Submission date |
Dec 22, 2020 |
| Last update date |
Mar 17, 2022 |
| Contact name |
Clifford G. Tepper |
| E-mail(s) |
cgtepper@ucdavis.edu
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| Phone |
916-734-7195
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| Organization name |
UC Davis School of Medicine
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| Department |
Biochemistry and Molecular Medicine
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| Street address |
4645 2nd Avenue, Room 2300A
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| City |
Sacramento |
| State/province |
CA |
| ZIP/Postal code |
95817 |
| Country |
USA |
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| Platform ID |
GPL25719 |
| Series (2) |
| GSE163694 |
Integrated analyses reveal CHD4 mediating establishment and maintenance of latent KSHV chromatin [Capture Hi-C] |
| GSE163695 |
Integrated analyses reveal CHD4 mediating establishment and maintenance of latent KSHV chromatin |
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| Relations |
| BioSample |
SAMN17137052 |
| SRA |
SRX9717894 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4984003_iSLK_ORF57-KO_lytic_CHi-C.hic |
845.6 Mb |
(ftp)(http) |
HIC |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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