NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM5277474 Query DataSets for GSM5277474
Status Public on Dec 01, 2021
Title IgG_BC1_2
Sample type SRA
 
Source name human primary effusion lymphoma
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
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.
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.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
Description Illimuna next-generation sequencing (2x150bp, paired-end)
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.
 
Submission date May 03, 2021
Last update date Dec 03, 2021
Contact name Clifford G. Tepper
E-mail(s) cgtepper@ucdavis.edu
Phone 916-734-7195
Organization name UC Davis School of Medicine
Department Biochemistry and Molecular Medicine
Street address 4645 2nd Avenue, Room 2300A
City Sacramento
State/province CA
ZIP/Postal code 95817
Country USA
 
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
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap
External link. Please review our privacy policy.