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| Status |
Public on Dec 13, 2018 |
| Title |
HPV+ HNSCC cell line SCC-090 h3k9ac |
| Sample type |
SRA |
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| Source name |
SCC-090 h3k9ac
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| Organism |
Homo sapiens |
| Characteristics |
cell type: HPV+ HNSCC cell line
patient id: SCC-090
hpv integration: Integrated
hpv type: HPV16
age: NA
gender: NA
race: NA
smoking: NA
type of tobacco: NA
pack years: NA
alcohol use: NA
antibody: h3k9ac
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-DNA was prepared using recently developed SimpleChIP Enzymatic Chromatin IP Kit #9005 (Cell Signaling Technology) following manufacturer’s protocol with sample-specific adjustments in micrococcal nuclease and sonication steps. 10X phosphate buffered saline (PBS) pH 7.4 from Quality Biological Inc. was used wherever PBS is indicated. Samples were digested by both micrococcal nuclease and sonication. This process was additionally optimized and followed by gel electrophoresis to ensure uniform lysis of DNA across the genome. Samples were treated with 0.5 µl of micrococcal nuclease (Cell Signaling Technologies) per IP prep for 20 minutes (500 µL total reaction volume in 1.7 mL Eppendorf tube), followed by 30 seconds sonication on intensity #2 out of 10 (550 Sonic Dismembrator, Fisher Scientific) in iced-call water-bath. Sonicated samples were left for one minute on ice. This was repeated 10 times over a 15-minute period. Freshly iced water was replaced 3 times during the cycle. Cleared chromatin preparation was analyzed for the level of digestion and DNA concentration, using recommended IV step from the kit protocol. Only chromatin preps with the distribution of chromatin DNA ranging from 150 bp to 900 bp in size were used for the immunoprecipitation step. DNA concentration was analyzed with a NanoDrop 1000 (Thermo Scientific) using the nucleic acid application module. Equal amounts of chromatin were used per IP step with exceptional performance (XP®) monoclonal antibodies validated for ChIP application (Cell Signaling Technology). Rabbit monoclonal antibodies were added in particular dilution based on an optimized concentration evaluated across a wide variety of commercial monoclonal antibodies. A 1:50 dilution for H3K4me3 (9751), H3K9ac (9649), H9K9me3 (13969) antibodies and a 1:100 dilution for H3K27ac (8173) antibody were used to isolate DNA segments bound by individual histone modification. We used 1:50 diluted total H3 (4620) antibody as a positive control and 1:250 diluted Normal Rabbit IgG (2729) as a negative control. A 3527-5 Incubator Shaker (Lab-Line) was used during elution. ChIP-DNA was purified and measured following the ChIP kit protocol. The 1/50 portion (2%) of the same chromatin for each sample was used for DNA extraction skipping the antibody enrichment steps and was further used for sequencing as an input control.
ChIP-DNA for individual sample/antibody and their input controls were sonicated, end-repaired, and ligated to SOLiD P1 and P2 sequencing adaptors lacking 5' phosphate groups, using the NEBNext DNA Library Prep Set for SOLiD per the manufacturer's recommended protocol (NEB). Libraries were then nick-translated with Platinum Taq. ChIP-DNA was sequenced at the Experimental and Computational Genomics Core (ECGC) at Johns Hopkins University with a target sequencing coverage of approximately 45,000,000x and paired-end reads of 150 bp. Illumina CASAVA 1.8.2 was used to convert BCL files to FASTQ files using default parameters. Bowtie 2.2.1 was used to map paired-end reads to the hg19 human reference genome using default parameters and samtools 0.1.19 was used to convert, sort, and index SAM files. The count functionality IGVTools package was used to generate a tiled data file using default parameters. MACS (Model-based Analysis of ChIP-Seq algorithm, version 1.4.2) called ChIP-Seq peaks for each mark and each sample using the input DNA in that sample as a control. ChIP-Seq peaks were called significant if MACS modeled peak p-values are below a threshold of 0.000001, and these peaks were represented as genomic intervals. The cis-regulatory element annotation system (CEAS) was used to associate these genomic intervals with genes.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Illumina's CASAVA 1.8.2 was used to convert BCL files to FASTQ files (default parameters)
bowtie2.2.1 (default parameters) was used to map paired end reads to hg19
MACS1.4.2 was used to call peaks (default parameters)
Genome_build: hg19 (GRCh37)
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| Submission date |
Dec 12, 2018 |
| Last update date |
Dec 14, 2018 |
| Contact name |
Elana Judith Fertig |
| E-mail(s) |
ejfertig@jhmi.edu
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| Organization name |
Johns Hopkins University
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| Department |
Oncology Quantitative Sciences
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| Street address |
550 N Broadway, Suite 1101E
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21209 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE112021 |
Integrated Analysis of Whole-Genome ChIP-Seq and RNA-Seq Data of Primary Head and Neck Tumor Samples Associates HPV Integration Sites with Open Chromatin Marks (ChIP-Seq) |
| GSE112027 |
HPV |
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| Relations |
| BioSample |
SAMN10585724 |
| SRA |
SRX5126743 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3510063_2_h3k9ac_peaks.narrowPeak.gz |
370.5 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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