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| Status |
Public on Apr 03, 2014 |
| Title |
6.15_LSK_KO_H3K4me1 |
| Sample type |
SRA |
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| Source name |
sorted murine lin-/Sca-1/c-Kit in vitro cultured cells
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| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl/6.SJL congenic
cell type: LSK hematopoietic stem cells
treatment: 4-OHT 48 hrs
antibody/vendor/catalog#: H3K4me1/Abcam/8895
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| Treatment protocol |
LSK cells were incubated in HSC medium supplemented with 300-400 nM 4-OHT (Sigma) to induce maximal deletion without harming cell viability. 4-OHT was washed out of the culture at 24 hours and cells were incubated an additional 24 hours.
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| Growth protocol |
HSC medium (SFEM (StemCell Technology) plus 300 ng/mL rmSCF, 20 ng/mL rm IL-11, and 4 ng/mL rmFlt3L (R&D Systems)).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells in 96 wells were pooled and resuspended to 1 million/mL in phosphate-buffered saline (PBS) containing 1% formaldehyde and incubated 10 minutes, followed by centrifugation and resuspension in 50 mM glycine/PBS, a 10 minute incubation, then a PBS incubation for 10 minutes. All steps were performed at room temperature at a cell density of 1 million/mL. Fixed cell pellets were either processed immediately or stored at -80°C.To shear the chromatin, the cell pellet was resuspended in lysis buffer (Tris pH 7.5, EDTA 1 mM, SDS 1%, 1X protease inhibitor complex, Roche) at 5x104 cells per 20 µL. Low-retention surface barrier tips were used for all steps (CLP Neptune). Sonication was performed for 10 cycles (30 seconds with 30 seconds rest) using a Bioruptor UCD-200 (Diagenode Inc.). Sonicated chromatin was centrifuged at 13,000 x g at 4°C for 5 minutes and the supernatant was diluted 10-fold with 2X RIPA buffer (20 mM Tris pH7.5, 2 mM EDTA, 2% Triton X-100, 0.1% SDS, 0.2% sodium deoxycholate, 200 mM NaCl). For each ChIP reaction 200µL diluted chromatin incubated with 1 µg antibody overnight at 4°C then 7.5 µL each of protein A and protein G Dynabeads (Invitrogen), previously washed in 1X RIPA buffer, were added to each immunoprecipitation and incubated for additional 2 hours at 4°C. The bead:protein complexes were washed three times with 200 µL 1X RIPA buffer and once with 200 µL TE (10mM Tris pH 7.5, 1mM EDTA). Genomic DNA was eluted from the ChIP and input samples for 3 hours at 65°C in 300 µL elution buffer (20 mM Tris pH 7.5, 5 mM EDTA, 50 mM NaCl, 1% SDS, 50 µg/mL proteinase K) using an Eppendorf Thermomixer at 1,000 rpm. Samples were phenol/chloroform extracted, ethanol precipitated with 10 µg each linear acrylamide and glycogen then centrifuged at 13,000xg for 20 minutes at 4°C. Pellets were air dried and resuspended in 15 µL TE containing 0.1 mM EDTA.
The library for sequencing was constructed using Ovation Ultralow DR Multiplex System 1-8 according to manufacturer's instructions (Nugen, San Carlos CA). Libraries were sequenced using HIseq-2000 (Illumina, San Diego, CA) to obtain 50 bp long reads. Half of the input or of the ChIP sample was used to prepare sequencing libraries with specific barcodes for individual ChIP sample according to the manufacturer’s instructions (Ovation Ultralow Library Systems, NuGEN). DNA concentration in each ChIP-library was measured using Qubit Fluorometer (Invitrogen) and 10 nM DNA from each library was used for running next-generation multiplexed sequencing using 50 bp single-end reads on a HiSeq-2000 instrument (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Debarcoding of the multiplex runs was performed using Unix shell scripts.
Tags were mapped to the mouse genome (mm9) using bowtie v0.12.7 with parameters -v 2 -m 1 -S --best --strata
Peak identification was performed with MACS v1.3.7.1 using default parameters
bedgraph files were created using Homer (Heinz et al., 2010) using -auto parameter converted into bigwig (bedGraphToBigWig) and visualized on UCSC (Kent et al., 2002) genome browser as custom tracks
the ChIP-diff method (Xu et al., 2008) was used to identify DNA regions with significant histone modification enrichment. The method divides the whole genome into bins with equal size of 1kb and compares the tags (reads) between two samples (e.g. S1 versus S2) to calculate the posterior probabilities (P1, P2 and P0 with P1+P2+P3=1) of each bin to be S1-enriched, S2-eriched or non-differential between S1 and S2. S1-enriched bins are defined as those with 1.5-fold tag enrichment in S1 versus S2, and with a posterior probability P1>0.95. We applied ChIP-diff to compare Mll1∆/∆ with control to further categorize bins into Mll1∆/∆-enriched, control-enriched and non-differential bins. Finally, enriched bins in a sample within 1kb in distance were merged into regions to yield the enriched regions for that sample.
Genome_build: mm9
Supplementary_files_format_and_content: BIGWIG file with ChiP enrichment signal
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| Submission date |
Apr 02, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Patricia Ernst |
| E-mail(s) |
patricia.ernst@ucdenver.edu
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| Phone |
303-724-8804
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| Organization name |
University of Colorado
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| Department |
Pediatrics
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| Lab |
Heme/Onc/BMT
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| Street address |
12800 E. 19th Ave
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| City |
Aurora |
| State/province |
CO |
| ZIP/Postal code |
80045 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE56456 |
The histone methyltransferase activity of MLL1 is dispensable for hematopoiesis and leukemogenesis |
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| Relations |
| BioSample |
SAMN02716081 |
| SRA |
SRX507968 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1361804_6.15_LSK_KO_H3K4me1.bw |
79.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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