|
| Status |
Public on Apr 18, 2013 |
| Title |
ASF1a ChIP |
| Sample type |
SRA |
| |
|
| Source name |
ASF1a ChIP
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: HeLa
chip antibody: equimolar mixture of Ms mAbs MPH7, MPB1, MPD5 (Ambagala et al., 2009)
|
| Treatment protocol |
Adherent cells were washed with PBS and cross-linked using sequential treatment with EGS and formaldehyde. Cells were incubated in 1.5mM EGS (Sigma, E3257), PBS for 45 min at room temperature, then formaldehyde (Sigma, 252549) was added to the media to 1% final concentration and cells were incubated for 15 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.
|
| Growth protocol |
HeLa cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-Glutamine, 50 U Pen-Strep and incubated at 37°C in a humidified 5% CO2 atmosphere. Every 3-4 days the cells were passaged to maintain 40-90% confluence level
|
| 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 Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow 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 band isolated from an agarose gel 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.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
ChIP-seq of histone chaperone ASF1a
|
| Data processing |
Basecalls performed using CASAVA 1.6
Sequenced reads were mapped to hg18 whole genome using bowtie v0.12.7 with parameter -p 8 -m 1 --best and remaining default settings
Aligned reads normalised by library size, extended to 150bp and converted to bigWig format
USeq 7.1.2 was used to perform peak finding 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)
Genome_build: hg18
Supplementary_files_format_and_content: bed files represent aligned reads (one line per read) in UCSC bed format. bigWig files represent pileups of the reads at each base. 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
|
| |
|
| Submission date |
Mar 11, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter D. Adams |
| Organization name |
University of Glasgow, Beatson Institute for Cancer Research
|
| Street address |
Switchback Rd, Bearsden
|
| City |
Glasgow |
| ZIP/Postal code |
G61 1BD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE45024 |
Genome-wide maps of chromatin state for HIRA, UBN1 and ASF1a |
| GSE45025 |
Placing the HIRA histone chaperone complex in the chromatin landscape |
|
| Relations |
| SRA |
SRX248766 |
| BioSample |
SAMN01975343 |
| Named Annotation |
GSM1095933_asf1a.s_4_sequence.txt.hg18.bt.map.bed.extended.bigWig |
