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| Status |
Public on Mar 17, 2022 |
| Title |
CHD4_BcBL-1_1_CUT&RUN |
| Sample type |
SRA |
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| Source name |
BCBL-1
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| Organisms |
Homo sapiens; Human gammaherpesvirus 8 |
| Characteristics |
tissue: human primary effusion lymphoma
cell type: KSHV-positive human B cell lymphoma cells
cut&run antibody: CHD4 (CST, D4B7)
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| Growth protocol |
ORF57-Wt iSLK and ORF57-KO iSLK cells were cultured in complete DMEM supplemented with 10% FBS. Cells were treated with or without 1 µg/mL doxycycline for 28 h. BCBL-1, BC-1, and BC-3 cells were grown in RPMI 1640 medium supplemented with 15% FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed with PBS and wash buffer (20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine (Sigma, S2626) and proteinase inhibitor (Roche). After removing wash buffer, cells were captured on magnetic ConA beads (Polysciences), in presence of CaCl2. Beads/cells complexes were washed with digiton wash buffer (0.02% digitonin, 20 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine and 1x proteinase inhibitor) three times, and incubated with specific antibodies in 250 mL volume. After incubation, unbound antibody was washed by digitonin wash buffer 3 times. Beads were then incubated with the recombinant pAG-MNase, in 250 μl digitonin wash buffer (1.0 mg/mL final concentration) for an hour at 4°C with rotation. Unbound pAG-MNase was removed by washing with digitonin wash buffer 3 times. Pre-chilled 2 mM CaCl2 containing digitonin wash buffer (200 mL) was added to beads and incubate on ice for 30 min. The pAG-MNase digestion was halted by the addition of 200 μl 2× STOP solution (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 μg/ml RNase A, 50 mg/ml Glycogens). The beads were incubated/shaked at 37°C for 10 min in tube shaker at 500 rpm to release digested DNA fragments from the insoluble nuclear chromatin. The supernatant was collected after centrifuge (16,000 x g for 5 min at 4°C) and place on magnetic stand. DNA was extracted using NucleoSpin kit (Takara).
Sequencing libraries were prepared from 3 ng of CUT&RUN DNA with the Kapa HyperPrep Kit (Roche) according to the manufacturer’s standard protocol. Libraries were multiplex sequenced (2 x 150bp, paired-end) on an Illumina HiSeq 4000 sequencing system.
Other: CUT&RUN
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Base calling and quality scoring were performed with Real-Time Analysis (RTA2) software.
CUT&RUN reads were aligned to the GRCh38 (hg38) human reference genome and KSHV reference genome (Human herpesvirus 8 strain: GQ994935.1) with Bowtie2.
MACS2 was used for detecting peaks following the developer’s manual. The default settings with a minimum FDR (q-value) cut-off of 0.05 were used.
Genome_build: GRCh38 (hg38) + KSHV genome (Accession: GQ994935.1)
Supplementary_files_format_and_content: Processed data are in bigWig format.
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| Submission date |
Dec 13, 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) |
| GSE163098 |
Integrated analyses reveal CHD4 mediating establishment and maintenance of latent KSHV chromatin [CUT&RUN] |
| GSE163695 |
Integrated analyses reveal CHD4 mediating establishment and maintenance of latent KSHV chromatin |
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| Relations |
| BioSample |
SAMN17071859 |
| SRA |
SRX9679423 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4972033_CHD4_BcBL-1_1.sorted.bw |
150.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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