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| Status |
Public on Jun 04, 2016 |
| Title |
A3 ZFP57 ChIP-seq |
| Sample type |
SRA |
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| Source name |
A3
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| Organism |
Mus musculus |
| Characteristics |
cell type: Embryonic stem cells
strain: 129X1/SvJ
chip antibody: ZFP57 (Abcam, ab45341)
genotype/variation: wild type
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| Growth protocol |
Embryonic stem cells were cultured under standard feeder-free conditions on gelatinized tissue culture dishes with media containing DMEM (EuroClone ECM0101L) supplemented with 100 µM 2-mercaptoethanol (Sigma), 1 x non-essential amino acids (only for A3), 1 mM sodium pyruvate, 2 mM L-glutamine, 1 x penicillin-streptomycin, 10% (15% for E14) foetal calf serum (HyClone) and 103 U/ml leukemia inhibitory factor (LIF, Millipore) at 37 °C under an atmosphere of 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed on formaldehyde cross-linked chromatin isolated from cells grown on 10 cm dishes to ~80% confluency. Briefly, the cells were detached by adding 0.05% trypsin at 37 °C for 3 min. Formaldehyde was added to the cells resuspended in PBS at a final concentration of 1% and the cells were incubated at room temperature for 15 min with shaking. The reaction was stopped by addition of glycine to a final concentration of 0.125 M. Approximately 3x107 cells were washed twice in ice-cold PBS, centrifuged and resuspended in lysis buffer 1 (50 mM HEPES pH 8, 10 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% NP-40 and 0.25% Triton X-100) for 90 min at 4 °C. Isolated nuclei were lysed in lysis buffer 2 (10 mM Tris–HCl pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) for 60 minat 4 °C. The chromatin was sheared in sonication buffer (10 mM Tris–HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% N-lauroylsarcosine) to an average size of 100–400 bp using the S220 Focused-ultrasonicators (Covaris). For each IP, 100 μg of sonicated chromatin were diluted in a final volume of 600 μl with sonication buffer and pre-cleared with 30 µl protein A/G agarose beads (SantaCruz) for 4 h at 4 °C on a rotating wheel. Anti-ZFP57 antibody (8 μg, Abcam ab45341), anti-KAP1 antibody (7 μg, Abcam ab10483) and anti-Histone H3K9me3 (7 μg, Abcam 8898) or rabbit IgG were added to the pre-cleared chromatin and incubated overnight at 4 °C on a rotating wheel. Chromatin was precipitated with 30 μl protein A/G agarose beads for 4 h at 4 °C with rotation. The beads were then washed five times with 500 μl RIPA buffer (10 mM Tris–HCl pH 8.0, 140 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 1% Triton X-100and 0.1 % SDS) and once with each of the following buffers: WASH buffer (50 mM HEPES, 0.5% sodium deoxycholate, 1% Triton X-100, 1 mM EDTA, 500 mM NaCl and 0.2% NaN3), LiCl buffer (0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA and 10 mM Tris pH 8) and TE buffer (10 mM Tris pH 8, 1 mM EDTA). The bound chromatin was eluted in 100 μl TE buffer. Crosslinks were reversed by incubation O/Nat 65°C after addition of 1 μl RNAse cocktail (Ambion) and 2h at 50°C after addition of 2.5μl SDS 20% + 2.5μl 20mg/ml proteinase K (Sigma). The DNA was extracted by using the QIAquick Gel Extraction Kit (Qiagen). Immunoprecipitated or 1% input DNAswere analysed by real-time PCR using SBYRGreen PCR Master Mix (Bio-Rad) on a C1000 Thermal Cycler (Bio-Rad).
Two nanograms of DNA from immunoprecipitated and input chromatin were used for Illumina library preparation. Libraries were generated by using the NuGen Ovation Ultralow Library System Kit.
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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 |
Basecalls and adaptor trimming was performed using Illumina Pipeline Casava (v1.8.2).
Reads were aligned to the NCBI37/mm9 genome assembly using Bowtie (v0.12.7) with uniquely mapping options.
Genomic reads coverage was obtained using SAMtools (v0.1.18) and BEDTools suite (v2.14.3-1).
Peak-calling was performed using MACS algorithm implemented in SeqMonk software (v0.24.1).
Genome_build: (NCBI37/mm9)
Supplementary_files_format_and_content: BedGraph file reports normalized RPM (reads per Million reads) scores of genomic coverage. Bed file contain genomic coordinates of called peaks.
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| Submission date |
Feb 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Marco Cammisa |
| E-mail(s) |
cammisamarco.py@gmail.com
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| Organization name |
Institute of Genetics and Biophysics "A. Buzzati Traverso" (IGB-ABT)
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| Street address |
Via Castellino Pietro, 111
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| City |
Naples |
| State/province |
Naples |
| ZIP/Postal code |
80131 |
| Country |
Italy |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE77440 |
ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells (ChIP-seq) |
| GSE77444 |
ZFP57 maintains the parent-of-origin-specific expression of the imprinted genes and differentially affects non-imprinted targets in mouse embryonic stem cells |
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| Relations |
| BioSample |
SAMN04450700 |
| SRA |
SRX1556082 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2051867_A3_ZFP57_ChIP-seq.bed.gz |
10.8 Kb |
(ftp)(http) |
BED |
| GSM2051867_A3_ZFP57_ChIP-seq.bedgraph.gz |
205.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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