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| Status |
Public on Aug 16, 2022 |
| Title |
Msh2_rep2 |
| Sample type |
SRA |
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| Source name |
E14
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14
cell type: mESCs
chip antibody: Streptavidin beads for MSH2 Bio-Tag ChIP + MSH2 transfected (Dynabeads MyOne streptavidin T1, ref 65601, Invitrogen)
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| Growth protocol |
mESCs were cultured under standard feeder-free conditions on gelatinized tissue culture dishes and maintained in DMEM (Gibco, Thermo Fisher Scientific) supplemented with 100 uM 2-mercaptoethanol (Sigma), 1 mM sodium pyruvate, 2 mM L-glutamine, 1x penicillin–streptomycin, 15% fetal calf serum (HyClone) and 103 U/ml leukemia inhibitory factor (LIF, Millipore).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP for the analysis of biotin tagged-MSH2, MSH6, 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 approximately 3 × 107 cells resuspended in Phosphate buffered saline (PBS) at a final concentration of 1% and the cells were incubated at room temperature for 10 min with shaking. The reaction was stopped by addition of glycine to a final concentration of 0.125 M. 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 10 min at 4°C. Isolated nuclei were lysed in lysis buffer 2 (10 mMTris–HCl pH 8.0, 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) for 10 min at 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-ultrasonicator (Covaris). Sonicated chromatin were diluted in sonication buffer. Chromatin for MSH6, ChIP was pre-cleared with 30 μl protein A/G agarose beads (SantaCruz) for 4 h at 4°C on a rotating wheel. Streptavidin beads (Dynabeads MyOne streptavidin T1, ref 65601, Invitrogen), anti-MSH6 (Santa Cruz sc-137015) or rabbit/mouse IgG were added to the pre-cleared chromatin and incubated overnight at 4°C on a rotating wheel.Chromatin for MSH6 ChIP 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 mMTris pH 8) and TE buffer (10 mM Tris pH 8, 1 mM EDTA). The bound chromatin was eluted in 100 μl TE buffer. Cross-links were reversed by incubation at 1 h at 37°C after addition of 1 μl RNAse cocktail (Ambion) and O/N at 60°C after addition of 2.5 μl SDS 20% + 2.5 μl 20 mg/ml proteinase K (Sigma). The DNA was extracted by using the QIAquick Gel Extraction Kit (Qiagen). Immunoprecipitated or 1% input DNAs were analysed by real-time PCR using SBYR Green PCR Master Mix (Bio-Rad) on a CFXCONNECT Thermal Cycler (Bio-Rad).
For ChIP-seq analysis, two nanograms of DNA from immunoprecipitated and input chromatin were used for Illumina library preparation. Libraries were generated and sequenced at IGA Technology Services (Italy), by using the NuGen Ovation Ultralow Library System v2 Kit and 150 bp paired-end sequencing on the Illumina NovaSeq6000 platform (Illumina, San Diego, CA)
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
First, we checked the quality of the base call and the presence of the adapters in the raw data. There were no bad quality base calls and adapter contaminations, so, we aligned the reads against the Mus musculus genome (mm10) using Bowtie2 short read aligner v2.3.5.1 with default parameters (Langmead and Steven L. Salzberg, 2012). We removed duplicate reads using Picard MarkDuplicates v2.22.9 [http://broadinstitute.github.io/picard/] and we only used uniquely mapped reads for the rest of the analysis.
The DNA binding profiles of ZFP57 and KAP1 in the E14 ESCs were obtained from the GSE77744 dataset. The coordinates were converted into the mm10 genome by using CrossMap Python script (Zhao et al., 2014).
For the visualization in UCSC, we used tracks normalized by reads per milion (RPM) generated by the GenomeCoverageBed tools of the BEDtools suite v2.292 (Quinlan and Ira M. Hall, 2010). To define the enriched regions, we used MACS2 algorithm with PE and –broad parameters (Zhang et al., 2008).
We considered only the intersection of the peaks of the two replicates for the rest of the analysis, obtained using the intersectBed function of BEDtools suite v2.292.
Assembly: mm10
Supplementary files format and content: bigWig
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| Submission date |
May 28, 2022 |
| Last update date |
Aug 16, 2022 |
| Contact name |
Francesco Cecere |
| E-mail(s) |
francesco.cecerengs@gmail.com
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| Phone |
3888903265
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| Organization name |
University of Campania "Luigi Vanvitelli"
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| Department |
DiStABiF
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| Lab |
Riccio
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| Street address |
Via A. Vivaldi, n. 43
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| City |
Caserta |
| State/province |
Caserta |
| ZIP/Postal code |
81100 |
| Country |
Italy |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE205041 |
The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex (ChIP-Seq) |
| GSE205043 |
The mismatch-repair proteins MSH2 and MSH6 interact with the imprinting control regions through the ZFP57-KAP1 complex |
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| Relations |
| BioSample |
SAMN28728449 |
| SRA |
SRX15492538 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6204475_msh2.rep2.bw |
264.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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