GSM3510063: HPV+ HNSCC cell line SCC-090 h3k9ac; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
Histone
Antigen
H3K9ac
Cell type
Cell type Class
Others
Cell type
Head and neck squamous cell carcinoma
NA
NA
Attributes by original data submitter
Sample
source_name
SCC-090 h3k9ac
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
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_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.