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| Status |
Public on Jun 25, 2013 |
| Title |
H3K4me2_Gr1dim_WT |
| Sample type |
SRA |
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| Source name |
Gr1dim Mac1+ granulocytic progenitor cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: Bone marrow derived Gr1dim Mac1+ granulocytic progenitor cells
strain: C57BL/6/SV129
genotype/variation: Lsd1fl/fl (wild type)
chip antibody: H3K4me2
antibody vendor/catalog/lot#: Millipore, 07-030, lot 1570816
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| Treatment protocol |
Both, Lsd1fl/fl and Lsd1fl/fl Mx1Cre mice were injected with three doses of high molecular weight p(I:C) (Invivogen, San Diego, USA) at a concentration of 12.5µg/g body weight. Forty million Gr1dim Mac1+ were cross-linked with 1% formaldehyde for 10 min at room temperature. Crosslinking was stopped with 125mM glycine for 5 minutes and cells were rinsed twice with 1X PBS. Cells were resuspended, lysed in lysis buffer, and sonicated to solubilize and shear cross-linked DNA. The samples were sonicated using a Heat Systems Ultrasonic Processor with a micro tip (settings: duty cycle 60%, output 4.5, 1 sec pulse, 30 sec. 6-10 times with at least 60 second pause between pulse sets) while suspended in a sonication buffer containing 10mM Tris-HCl pH7.4, 300mM NaCl, 1mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% NaDOC. Samples were kept on an ethanol ice bath at all times. The resulting whole cell extract was split into chromatin aliquots equivalent to 10x106 of input cells and incubated overnight at 4°C with approximately 5 µg of the appropriate antibody. Protein A Dynabeads (Invitrogen) were used for collection of chromatin. Beads were washed 2X with 10mM Tris-HCl pH 7.4, 1mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% NaDOC; 2X with 10mM Tris-HCl pH 7.4, 300mM NaCl, 1mM EDTA, 0.1% SDS, 1% Triton X-100, 0.1% NaDOC; 2x with 10mM Tris-HCl pH 8.0, 250mM LiCl, 2mM EDTA, 0.5% NP40, 0.5% NaDOC and 2X with TE. Bound complexes were eluted from the beads (50 mM Tris-Hcl, pH 8.0, 10 mM EDTA and 1% SDS) by heating at 65°C for 1 hour with occasional vortexing. Crosslinking was reversed by overnight incubation at 65°C. Ten percent of input DNA was also treated for crosslink reversal.
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| Growth protocol |
Freshly isolated primary bone marrow cells, the cells have NOT been cultured in vitro.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody or no antibody for Input samples. ChIP DNA was quantified by Qubit assay HS kit. For ChIP-Seq analysis using the HeliScope™ Single Molecule Sequencer, ChIP DNA was processed for 3’ polyA tailing, followed by 3’ ddATP-blocking as described previously (Hart et al., 2010; Thompson and Steinmann, 2010). Processing samples by ligation, amplification and size selection are not required by Helicos sequencing. Briefly, 6-10 ng of ChIP DNA or input DNA was used in the 3’ polyA tailing reaction in 14.8 μl containing 2 μl of 2.5 mM CoCl2, 2 μl of 10 x terminal transferase buffer and nuclease-free water. The reaction mixture was denatured in 95oC for 5 min, followed by rapid cooling in ice water slurry. Then 1 U of terminal transferase, 4 μl of 50 μM dATP and 0.2 μl of NEB BSA were added to the mixture. Samples were incubated in a thermocycler at 37°C for 1 h, 70°C 10 min, followed by denaturing at 95°C for 5 min and rapid cooling in ice water slurry. For 3’ ddATP-blocking, 0.5 μl of 200 μM ddATP, 1 μl of 10 x terminal transferase, 1 μl of 2.5 mM CoCl2, 1 U of terminal transferase, and 6.5 μl of nuclear-free water were added to the above reaction. Samples were incubated in a thermocycler at 37°C for 1 h and 70°C for 20 min. Then 2 picomoles of carrier oligonucleotide were added to the reaction, and samples (=H3K4me2-ChIP and H3K4me3-ChIP from wild type and Lsd1 knockout Gr1dim Mac1+ cells + matching input control) were hybridized to the HelicosTM flow cells for sequencing at the Dana-Farber Cancer Institute (DFCI) Molecular Biology Core Facility. Sequencing reads were aligned to mouse genome assembly mm9 (NCBI Build 37) using Helisphere software with default parameters. Only the reads that were uniquely aligned to reference genome with read alignment score higher than 4.5 were retained for further analysis.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Helicos HeliScope |
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| Description |
FACS-purified primary Gr1dim Mac1+ bone marrow granulocyte progenitor cells
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| Data processing |
Basecalls performed using Solexa pipeline for data generated by HiSeq 2000 or Helicos pipeline for data generated by Helicos Heliscope.
Sequencing reads were aligned to mouse genome assembly mm9 using Illumina software pipeline (data generated by HiSeq 2000) or Helisphere (data generated by Helicos).
Only ChIP-seq reads that aligned to exactly one location in the reference mouse genome (mm9) were retained for downstream data analysis.
Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/).
The wig files were generated by a moving window of size 200bp. The tag count in the windown was further normalized in unit RPKM (# read per kb per million total reads) for ChIP-seq data generated by HiSeq 2000.
Genome_build: mm9
Supplementary_files_format_and_content: Mapped read bed file, wig file, and peak bed file
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| Submission date |
Aug 28, 2012 |
| Last update date |
Jun 25, 2013 |
| Contact name |
Marc A. Kerenyi |
| Organization name |
Boston Children's Hospital and Dana Farber Cancer Institute
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| Department |
Hematology / Oncology
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| Lab |
Stuart Orkin Laboratory
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| Street address |
1 Blackfan Street - Karp Research Building
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| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL14759 |
| Series (1) |
| GSE40440 |
Histone Demethylase Lsd1 is Required to Repress Hematopoietic Stem and Progenitor Cell Signatures During Blood Cell Maturation |
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| Relations |
| BioSample |
SAMN02197280 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM994232_H3K4me2_WT_binsize_200_stepsize_50_fixedstep.wig.gz |
5.0 Mb |
(ftp)(http) |
WIG |
| GSM994232_K4me2_wt_peaks.bed.gz |
214.2 Kb |
(ftp)(http) |
BED |
| GSM994232_wt_H3K4me2_4.3.garand.bed.gz |
32.1 Mb |
(ftp)(http) |
BED |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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