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Status |
Public on Dec 01, 2021 |
Title |
IgG_BC1_2 |
Sample type |
SRA |
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Source name |
human primary effusion lymphoma
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Organisms |
Homo sapiens; Human gammaherpesvirus 8 |
Characteristics |
cell line: BC-1 cell type: KSHV-positive human B cell lymphoma cells cut&run antibody: non-specific IgG
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Growth protocol |
BCBL-1, (TREx)BCBL-1, and BC-1 cell lines were cultured in RPMI 1640 medium supplemented with 15% FBS. The cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2 (v/v). Mycoplasma contamination was routinely tested by using a PCR-based assay.
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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 digitonin 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 the NucleoSpin & PCR kit (Takara Bio, Kusatsu, Shiga, Japan). 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.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
Illimuna next-generation sequencing (2x150bp, paired-end)
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Data processing |
Library strategy: CUT&RUN 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 |
May 03, 2021 |
Last update date |
Dec 03, 2021 |
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 |
GPL30065 |
Series (2) |
GSE173724 |
Targeting MYC Transcription with Small Peptide Derived from KSHV Transactivator (CUT&RUN) |
GSE173725 |
Targeting MYC Transcription with Small Peptide Derived from KSHV Transactivator |
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Relations |
BioSample |
SAMN18977381 |
SRA |
SRX10754403 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5277474_IgG_BC1_2.sorted.bw |
149.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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