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| Status |
Public on Dec 02, 2010 |
| Title |
cells.H3K4me3.IP.rep1 |
| Sample type |
SRA |
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| Source name |
Cells isolated from the spleen of leukemic blasts-injected mice, H3K4me3 ChIP
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| Organism |
Mus musculus |
| Characteristics |
strain: 129/SvEv
tissue: spleen
sample type: cells
antibody: anti-H3K4me3
antibody vendor: Active Motif
antibody catalog #: 39159
antibody lot #: 01609004
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| Treatment protocol |
None.
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| Growth protocol |
Leukemic blasts were isolated from APL transgenic mice and intravenously injected (1 x 10^6 cells) into syngeneic recipient mice to induce secondary leukemias (Minucci, S. et al. Blood 100, 2989-2995 (2002)) and establish a massive splenomegaly.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin preparation: Rehydrated formaldehyde-fixed and paraffin-embedded (FFPE) spleen sections (tissue) and APL leukemic cells isolated from spleens and cross-linked ex vivo (cells) were incubated in 0.5 ml permeabilization buffer (1xTBS; 0.5% Tween20; 1mM PMSF; 10 ug/ml RNAse A) for 30 min at RT in a rotating platform. After centrifugation at 18,000g for 5 min at +4°C, samples were resuspended in 200 ul digestion buffer (50mM Tris-HCl, pH 7.4; 0,32M sucrose; 4mM MgCl2; 1mM CaCl2; 0.1mM PMSF). FFPE-derived samples were partially fragmented through mild sonication, 3 times x 30 sec at 50W using a Labsonic L sonicator (B. Braun Biotech International) and then digested for 1 min at 37°C with micrococcal nuclease (USB - N.70196Y) at the final concentration of 1U/10ug of chromatin (based on spectrophotometric optical density measured at 260 nm). After centrifugation at 18,000g for 5 min at +4°C, samples were resuspended in 200 ul sonication buffer (1xTBS; 0.1% SDS; 1 mM Na2EDTA pH 8.0) and further fragmented 3 times x 30 sec (in ice-cold bath) either at 100W (FFPE samples) or 50W (Cells). After centrifugation at 8,000g for 5 min at RT, the first supernatant was collected (volume of approximately 170 ul). The pellets were washed once with 50 ul sonication buffer, vortexed for 5 sec, centrifuged again to obtain the second supernatant (to reach a final volume of approximately 220 ul).
Chromatin immunoprecipitation assay (PAT-ChIP and Cells-ChIP): Chromatin was quantitated fluorimetrically by Qubit Invitrogen, according to the manufacturer's instructions. Immunoselection of chromatin was carried out in ChIP buffer (30mM Tris-HCl, pH 7.4; 50mM NaCl; 5mM Na2EDTA; 0.1mM PMSF) in a final volume of 300 ul, using 260-600 ng of chromatin for each assay (dependent on either the amount of chromatin extracted from FFPE samples in each experiment and the number of ChIP assays to perform) and incubated 16 hr at +4°C in a rotating platform with the desired antibody (Active Motif rabbit anti-histone H3 trimethyl Lys4 pAb or Upstate rabbit anti-trimethyl-histone H3 (Lys27) pAb) at a previously determined optimal amount. 40ul of 50% v/v slurry rec-Protein G-Sepharose 4B Conjugate (preincubated 16 hours at +4°C with 1mg/ml of BSA in ChIP buffer - Zymed) were added to each ChIP assay and incubated for 3 hr at +4°C. After centrifugation at 2,000g, for 5 min at +4°C, pellets were washed sequentially with 10 ml of washing buffer A (50mM Tris-HCl, pH 7.4; 1% TritonX-100; 50mM NaCl; 5mM Na2EDTA; 0.1mM PMSF), 10 ml of washing buffer B (50mM Tris-HCl, pH 7.4; 1% TritonX-100; 100mM NaCl; 5mM Na2EDTA; 0.1mM PMSF), and 10 ml of washing buffer C (50mM Tris-HCl, pH 7.4; 1% TritonX-100; 150mM NaCl; 5mM Na2EDTA; 0.1mM PMSF). Elution was carried out by adding 200ul of elution buffer (1xTE/1%SDS) and incubating for 30 min at RT in a rotating platform. After centrifugation at 1,200g for 5 min at RT, the supernatant was saved and the elution repeated to obtain a final volume of 400ul (bound fraction).
DNA isolation: Decrosslinking was performed through an overnight incubation at 65°C in elution buffer/0.2M NaCl followed by a digestion with 80ug/ml proteinase K (3hr at +45°C). DNA was isolated by sequential extractions with one-third volume of phenol:chloroform (1:1), one volume of phenol:chloroform (1:1), one volume of chloroform and performing a centrifugation at 6,000g for 10 min at RT between each step of extraction. DNA was precipitated adding 1/10th volume of 5M sodium acetate, pH 5.2, 20ug of linear poly-acrylamide (LPA), 2.5 volumes of 100% ethanol (overnight at -20°C). After centrifugation at 3,000g for 30 min at +4°C, DNA pellets were washed once in 1 ml of ice-cold 70% ethanol solution, centrifuged, air dried and resuspended in 50ul of TE buffer (stored at -20°C).
Tagged libraries were produced according to Illumina's instructions from 7-10 nanograms of the bound and the input DNA, and as described by Schweiger, M.R. et al. Plos One 5, e5548 (2009).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Chromatin IP against H3K4me3.
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| Data processing |
Alignment: Sequence reads were mapped to the mouse mm9 genome using Bowtie (PMID: 19261174; Illumina pipeline version 1.3). All reads that map uniquely to the genome with two or fewer mismatches were retained.
Peaks: Analyses were performed using MACS (PMID: 18798982) with a p-value threshold of 10^-5, a bandwith of 300bp and the mfold parameter set to 16. All ChIPs were normalized against the corresponding input DNA. The peaks are in the *peaks.bed files.
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| Submission date |
Apr 21, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Iros Barozzi |
| E-mail(s) |
iros.barozzi@meduniwien.ac.at
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| Organization name |
Medical University Vienna
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| Street address |
Borschkegasse 8a
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| City |
Vienna |
| ZIP/Postal code |
1090 |
| Country |
Austria |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE21449 |
PAT-ChIP-Seq: a novel tool for the epigenetic profiling of formaldehyde-fixed and paraffin-embedded (FFPE) pathology samples |
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| Relations |
| SRA |
SRX028520 |
| BioSample |
SAMN00011917 |
| Named Annotation |
GSM536093_cells_H3K4me3_IP.rep1.bed.gz |
| Named Annotation |
GSM536093_cells_H3K4me3_IP_rep1_vs_input_peaks.bed.gz |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM536093_cells_H3K4me3_IP.rep1.bed.gz |
92.5 Mb |
(ftp)(http) |
BED |
| GSM536093_cells_H3K4me3_IP_rep1_vs_input_peaks.bed.gz |
278.3 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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