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| Status |
Public on Nov 26, 2014 |
| Title |
H4K16ac Prolif ChIP Rep2 |
| Sample type |
SRA |
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| Source name |
Fibroblasts
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| Organism |
Homo sapiens |
| Characteristics |
cell line: IMR90
chip antibody: H4K16ac (Abcam ab109463)
cell state: proliferating
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| Treatment protocol |
Cells were harvested by trypsinization and precipitated by centrifugation. Cells pellets were resuspended in 1% formaldehyde in DMEM and incubated for 5 more minutes. Cross-linking was quenched by adding 1/10 of volume of 1.25 M glycine (125mM final) and incubating for 5 min at room temperature. Media was aspirated and cells were rinsed twice with PBS, scraped into ice-cold PBS supplemented with protease inhibitors (Sigma, P8340) and collected by centrifugation (5 min at 200g, 4°C). Cell pellets were snap frozen in dry ice with ethanol and kept at -70°C before use. For HIRA ChIP IMR90 cells were grown to 80%–90% confluency in 15 cm plates. Three plates (about 20 million cells) were washed with phosphate buffered saline (PBS) and scraped into 6 ml of 10 mM Tris, 30 mM NaCl, 0.1% (v/v) NP40, 3 mM MgCl2 pH 7.5 supplemented with protease (Sigma, P8340) and phosphatase (Sigma, P5726) inhibitor cocktails. Following 10 min incubation on ice the suspension was centrifuged (3 min at 300 g, 4°C) to produce nuclear pellet. This pellet was resuspended in 1ml of benzonase buffer (50 mM Tris pH 7.5, 300 mM NaCl, 0.5% (v/v) NP40, 2.5 mM MgCl2) with protease and phosphatase inhibitors followed by addition of 125 U of benzonase (Sigma, E1014). The lysate was thoroughly mixed and incubated on ice for 30 min. Supernatant was recovered by centrifugation (5 min at 13000 g, 4°C), separated from the pellet and diluted with 1 ml of dilution/stop digestion buffer (50 mM Tris pH 7.5, 100 mM NaCl, 0.5% (v/v) NP40, 15 mM EDTA, protease and phosphatase inhibitors). The resulting lysate was used as chromatin preparation for ChIP.
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| Growth protocol |
IMR90 cells were cultured in DMEM supplemented with 20% (v/v) FBS, 2 mM L-Glutamine, 50 U/ml Pen-Strep and incubated at 37°C in a humidified 5% CO2 and 3% O2 atmosphere. Every 4-5 days the cells were passaged to maintain 40%–90% confluence level until they stopped proliferating through Replicative senescence or oncogene induced senescence
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cross-linked cells were solubilized by ultrasound treatment (Bioruptor Diagenode) to generate DNA fragmentation in 150-300 bp range. Protein-DNA complexes were isolated using corresponding antibody pre-immobilized on Dynabeads (Invitrogen).
Libraries were prepared according to NEB's instructions accompanying the NEBNext® ChIP-Seq Library Prep Master Mix Set for Illumina®(E6240S). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were isolated using Agencourt AMPure XP beads (Beckman Coulter) and PCR-amplified using Illumina primers. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
ChIP-seq of H4K16ac in proliferating IMR90 cells
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| Data processing |
Basecalls performed using CASAVA 1.8.2
Sequenced reads were trimmed using trim galore v0.3.0 with parameter --quality 20 and remaining default settings
Sequenced reads were mapped to hg19 whole genome using bowtie v2.1.0 with parameter -p 15 and remaining default settings
Multimapped reads were removed using samtools 0.1.16 and duplicate reads removed using picard tools v1.98 mark duplicates
Aligned reads normalised by library size, extended to 150bp and converted to bigWig format
USeq 8.6.0 was used to perform HIRA peak finding (normalised to input) with the following steps. BED files converted to Useq Point Format (Tag2Point). Satellite Repeat regions were removed from the dataset (FilterPointData). ScanSeqs used to identify enriched windows (-w 200 -p 150 -f). Enriched Regions Extracted (EnrichedRegionMaker) with 5% FDR and 2 fold change (-i 2,4 -s 13,1)
SICER v1.1 was used to perform histone peak finding (H3.3 was normalised to input, H4K16ac to H4) using redundancy threshold of 1, window size of 200bp, fragment size of 150bp, effective genome fraction of 0.75, gap size of 200bp and R of 0.01.
Differential binding sites for H4K16ac were identified using Diffbind v1.8.3 with the following steps. Reads were counted using an insert length of 150bp and scored using DBA_SCORE_READS_MINUS. Enriched regions were identified using the full library size, DBA_DESEQ method and remaining defaults.
Genome_build: hg19
Supplementary_files_format_and_content: BigWig files were normalised by library size and represent the number of reads at a given genomic location per million mapped reads and generated using UCSC wigToBigWig. peak files were generated with USeq and thresholded at 5% FDR and 2 fold change cutoffs
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| Submission date |
Mar 27, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Adams |
| Organization name |
University of Glasgow, Beatson Institute for Cancer Research
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| Street address |
Switchback Rd, Bearsden
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| City |
Glasgow |
| ZIP/Postal code |
G61 1BD |
| Country |
United Kingdom |
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| Platform ID |
GPL10999 |
| Series (1) |
| GSE56307 |
Histone chaperone HIRA orchestrates H4K16ac-decorated dynamic chromatin in senescent cells and is required for suppression of oncogene-induced neoplasia. |
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| Relations |
| BioSample |
SAMN02711335 |
| SRA |
SRX502817 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1358822_H4K16ac.Prolif.R2.bigwig |
84.7 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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