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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 01, 2012 |
| Title |
WT_H3K4me3_5 |
| Sample type |
SRA |
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| Source name |
Schneider's Drosophila Line 2; S2 cells
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| Organism |
Drosophila melanogaster |
| Characteristics |
cell type: S2
genotype/variation: WT
chip antibody: (rb) α-H3K4me3 (#528; Ali Shilatifard)
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| Treatment protocol |
We used 10 µg of dsRNA per 1.5 x 106 cells per 60 mm well (6 well plate) or 80 µg of dsRNA per 12 x 106 cells per T75 flask with 2.5 ml SFX medium(Thermo/HyClone #SH30278.02) and 1% Penicillin/Streptomycin (Invitrogen #15140) per 60 mm well and 20 ml SFX medium (1% Penicillin/Streptomycin) per T75 flask. dsRNA was mixed with the corresponding volume of S2 cells (0.6 x 106 S2 cells/ml) and distributed in either 60 mm wells or T75 flasks. RNAi-mediated knockdown was performed for 5 days. MEFs are homozygous wild-type or homozygous knock-outs of MLL1 or MLL3 as described in (Wang et al. Mol Cell Biol. 2009 November; 29(22): 6074–6085)
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| Growth protocol |
S2 cells were grown in SFX medium (Thermo/HyClone #SH30278.02) with 1% Penicillin/Streptomycin (Invitrogen #15140) at room temperature. Mouse embryonic fibroblasts were grown in DMEM with 10% FBS supplemented with glutamine.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
S2 cells (one T75 flask per histone antibody and two T75 flasks for other antibodies) were cross-linked in 1% formaldehyde (by adding 37% formaldehyde to medium) on a nutator for 15 min at room temperature (RT). Samples were quenched by adding 2.5 M glycine to a final glycine concentration of 225 mM and incubated on a nutator for 5 min at RT. Following centrifugation for 5 min at 2000 g at 4°C the supernatant was aspirated. The cell pellet was resuspended in 5 ml Orlando/Paro buffer (10 mM Tris HCl pH 7.5, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100, 0.5 mM DTT, protease inhibitors [Roche, complete, EDTA-free, Cat No. 05056489]) and centrifuged for 5 min at 2000 g at 4°C. The supernatant was aspirated and the wash step with Orlando/Paro buffer repeated for another two times. The cell pellet (~100 µl for one T75 flask) was resuspended in 1.5 ml RIPA buffer (25 mM Tris pH 7.5, 140 mM NaCl, 1% Triton X-100, 1 mM EDTA, 0.1% SDS, 0.1% Na-deoxycholate, 0.5% of N-lauroylsarcosine, 0.5 mM DTT, protease inhibitors [Roche, complete, EDTA-free, Cat No. 05056489]) per 100 µl of cell pellet and 1.5 ml samples sonicated for 15 min (50% on/off cycle, high) (Bioruptor, Diagenode) in 15 ml hard plastic tubes (Corning, 430055). Sonicated chromatin was centrifuged for 20 min at 14,000 rpm at 4° and the supernatant kept. 10 μl of the sonicated chromatin was kept for gel analysis (to check sizing pattern) and 50 μl was used as an Input control. Sizing samples were reverse cross-linked over night at 65ºC by adding 90 μl RIPA and 3 μl proteinase K (30 mg/μl) and input samples by adding 50 μl RIPA and 5 μl proteinase K and processed/purified in the same way as the ChIP samples (see below). The remaining chromatin was diluted two-fold with RIPA buffer (without N-lauroylsarcosine) and incubated over night with the respective antibody on a nutator at 4ºC. 60 μl of protein A agarose (Invitrogen, 15918-014) was washed in 5 ml RIPA buffer, centrifuged for 2 min at 1,000 rpm at 4ºC. The supernatant was aspirated, the chromatin sample added and incubated for 2 h on a nutator at 4ºC. After centrifugation for 2 min at 1,000 rpm at 4ºC the protein A agarose was transferred into a 1.5 ml tube, washed with 1 ml RIPA buffer, incubated for 5 min on a nutator at room temperature and centrifuged for 2 min at 2,500 rpm at 4ºC. The supernatant was aspirated and the previous washing steps repeated another five times. Elution was performed on a nutator at room temperature for 20 min with 300 μl elution buffer (0.1 M NaHCO3, 1% SDS) containing proteinase K (1 ml elution buffer and 5 μl proteinase K [30 mg/μl]) and the sample centrifuged for 2 min at 2,500 rpm at room temperature. The supernatant was kept and the elution step repeated. Elution fractions were pooled and reverse cross-linked over night at 65ºC. 1 µl of RNase A (R4642, Sigma) was added to reverse cross-linked samples followed by incubation for 1 h at 37°C. DNA was isolated with the Qiagen PCR purification kit (#28106) and eluted in 50 μl H2O and DNA concentration determined by Pico Green Assay (Invitrogen #P7581). Mouse embryonic fibroblasts (MEFs) ChIP was performed according to a previously described protocol (Wang et al. 2009). Briefly, approximately 10^7 MEFs for each assay were cross-linked with 1% formaldehyde and sonicated. 10 µg of antibody and 50 µl protein A agarose were used in the ChIP assays.
Libraries were prepared with the Illumina TruSeq RNA Sample Preparation Kit (#RS-122-2001,RS-122-2002) according to Illumina's instructions, adapted for multiplexing ChIP-seq samples by starting at the end repair step using 1/3 of the adapters. Libraries were sequenced on the Illumina Hi-Seq 2000 following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Chromatin Immunoprecipitation of H3K4me3 in WT_5
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| Data processing |
Base-calling and quality filtering, default settings, Illumina Casava 1.8
Reads were aligned to the fly genome (UCSC dm3), or mouse genome (UCSC mm9), using Bowtie v0.12.7, allowing unique reads only and up to three mismatches of the 50bp read length.
Enrichment for some ChIP-seq samples was determined by MACS v1.4.1 using default settings, the dm genome, and the associated input sample. The publication utilized a subset of peaks meeting the criteria (p < 1e-5 and fold change > 5, or FDR < 5%) but all peaks with p < 1e-5 are included in this submission.
Genome_build: UCSC dm3; UCSC mm9
Supplementary_files_format_and_content: txt.bam.bed file represents UCSC BED format for alignments.
Supplementary_files_format_and_content: peaks.tab file represents MACS v1.4.1 tab-delimited format for all enriched regions at p < 1e-5.
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| Submission date |
Oct 10, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Alexander (Garrett) Garruss |
| E-mail(s) |
[email protected]
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| Organization name |
Stowers Institute for Medical Research
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| Lab |
Shilatifard
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| Street address |
1000 East 50th Street
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| City |
Kansas CIty |
| State/province |
Missouri |
| ZIP/Postal code |
64110 |
| Country |
USA |
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| Platform ID |
GPL13304 |
| Series (1) |
| GSE41440 |
Enhancer-associated H3K4 mono-methylation by Drosophila Trithorax-related, the Drosophila homolog of Mll3/Mll4 |
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| Relations |
| SRA |
SRX193323 |
| BioSample |
SAMN01760708 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1017409_WT_H3K4me3_5.txt.bam.bed.gz |
79.9 Mb |
(ftp)(http) |
BED |
| GSM1017409_WT_H3K4me3_5_peaks.tab.gz |
95.2 Kb |
(ftp)(http) |
TAB |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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