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Status |
Public on Jan 07, 2016 |
Title |
H3K4me3-poly_rep2-A |
Sample type |
SRA |
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Source name |
HeLa cells
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Organism |
Homo sapiens |
Characteristics |
cell line: HeLa cells treatment: Paraformaldehyde fixation (1%, 30 minutes) chip antibody: H3K4me3 polyclonal Ref: Active motif 39159
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Treatment protocol |
40 million cells were sonicated in 500μL of Lysis Buffer (1% Na-deoxychlorate, 50mM TrisHCl pH8, 140mM NaCl, 1mM EDTA, 1% Triton X-100) containing 5-times diluted Protease Inhibitor Cocktail (PIC; Roche Diagnostic; 1 tablet solubilized in 10ml Lysis Buffer). Sonication was performed with a Bioblock Scientific instrument (Vibra Cell 75043; 40 cycles, 30s ON end 59s OFF; 38% power). Chromatin fragmentation was evaluated by agarose gel electrophoresis as follows: 20μL of sonicated chromatin was diluted with 20μL TE (10mM Tris-HCl pH 8, 1mM EDTA) and 5μL 5M NaCl was added. Diluted chromatin was incubated at 100°C for 30 min, centrifuged at 12,000 rpm at room temperature and the supernatant was loaded onto a 2% agarose gel.
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Growth protocol |
HeLa cells were grown in DMEM 1g/L glucose, 5% Fetal Calf Serum and 40μg Gentamicin to a density of 15–20 millions cells/15cm plates. Cells were fixed for 30min with paraformaldehyde (1% in PBS). Fixation was quenched with 0.2M glycine in PBS, then cells were washed three times with PBS, collected and stored at -80°C.
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Extracted molecule |
genomic DNA |
Extraction protocol |
25μL of ChIP-IT Protein G Magnetic Beads (ActiveMotif) were incubated with the antibody under evaluation (the amounts of antibody in use corresponded to that indicated by the supplier’s information) in presence of 100μL PIC-containing Lysis Buffer. After two hours at 4°C on a rotating shaker, chromatin from 3 million cells was added and the final vol- ume was adjusted to 500μL with PIC-containing Lysis Buffer and incubation on a rotating shaker was continued overnight at 4°C. The immunoprecipitated chromatin was recovered by magnetic bead separation, followed by multiple washing steps on a custom liquid handling platform (TECAN EVO75). Specifically, the washing is performed as follows: (1) Low salt washing (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM TrisHCl pH 8, 150mM NaCl); (2) High salt washing (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM TrisHCl pH 8, 500mM NaCl); (3) LiCl-washing (0.25M LiCl, 1% IGEPAL CA630, 1% Na-deoxycholate, 1mM EDTA, 10mM Tris pH 8) and (4) 1×TE washing. The immunoprecipitated chromatin was eluted and de-crosslinked in 100μL of elution buffer (1% SDS, 100mM NaHCO3, 250mM NaCl, 0.2mg/ml Proteinase K) and incubated for 4 hours at 65°C. The eluted chromatin was supplemented with 200μL H2O and 300μL phenol/chloroform/isoamyl alcohol (25/24/1) mix was added. After two extraction steps, the aqueous phase was subjected to ethanol precipitation in presence of 1μL GlycoBlue (Invitrogen; 15mg/ml). The precipitated material was re-suspended in 45μL H2O; 5μL was used for validation by quantitative PCR, the remaining 40μL was used for DNA library preparation. The DNA library preparation for massive parallel sequencing was performed according to standard procedures (NEXTFlex ChIP-Seq Kit (Biooscientific)) adapted to automation by our custom liquid handling platform (TECAN EVO75). Prior to DNA sequencing library preparation was monitored using a Tapestation (Agilent). Samples were sequenced on an Illumina HiSeq 2000 platform following manufacturer’s standard procedures and certified on the basis of the NGS-QC Generator concept (Mendoza-Parra et al; NAR 2013).
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina HiSeq 2000 |
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Description |
H3K4me3-poly_rep2.bw
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Data processing |
ChIP-seq and related datasets (IP and Input control) were quality certified with NGS-QC Generator tool (www.ngs-qc.org). Specifically, this methodology allows providing enrichment quality descriptors discretized in a scale ranging from “AAA” (Best) to “DDD” (worst). The quality scores reports associated to these datasets are available at : http://www.ngs-qc.org/certdb.php bigWig files were generated with a read elongation of 150nts and a resolution of 20nts Genome_build: hg19 Supplementary_files_format_and_content: bigWig files
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Submission date |
Jan 07, 2016 |
Last update date |
May 15, 2019 |
Contact name |
Marco Antonio Mendoza-Parra |
E-mail(s) |
marco@igbmc.fr
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Organization name |
IGBMC
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Street address |
1, rue Laurent Fries
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City |
Illkirch; Strasbourg |
ZIP/Postal code |
67400 |
Country |
France |
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Platform ID |
GPL11154 |
Series (1) |
GSE76618 |
Antibody performance in ChIP-sequencing assays: From quality scores of public datasets to quantitative certification |
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Relations |
BioSample |
SAMN04388077 |
SRA |
SRX1521241 |
Supplementary data files not provided |
SRA Run Selector |
Processed data are available on Series record |
Raw data are available in SRA |
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