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| Status |
Public on Mar 01, 2015 |
| Title |
Day4_H3K27me3_THF_Rep3 |
| Sample type |
SRA |
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| Source name |
Differentiated embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: Brg1f/f; Actin-CreER
timepoint: Day 4
antibody: anti-H3K27me3(Millipore 17-622)
brg1 levels: Normal
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| Treatment protocol |
On Day2, cultures were treated with either 200 nM 4-OHT in tetrahydrofuran (THF) or THF alone. 4-OHT induces CreER mediated deleted of Brg1 floxed alleles.
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| Growth protocol |
Mouse ES cells were cultured in feeder-free conditions under standard conditions. Mouse ES cells were aggregated into embryoid bodies (EB) and differentiated for two days in serum free media (Day2). Embryoid bodies were dissociated and reaggregated for 40 hours in the presence of VEGF, Activin A, and BMP4 to promote mesoderm differentiation (Day4).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Chromatin immunoprecipitation of histone modifications were performed according to the Young lab protocol (Lee et al. 2006) with minor modification in biological duplicate per experimental condition, except for H3K27me3 and BRG1-FLAG- ChIP-seq, which were performed in biological triplicate. Briefly, frozen pellets of cross-linked cells (10x106) were thawed in cold lysis buffer 1 (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1× protease inhibitors) and gently rocked at 4°C for 10 minutes in 15 mL conical tubes. Cells were pelleted at 1350 x g at 4°C and resuspended in cold lysis buffer 2 (10 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1× protease inhibitors) and gently rocked at 4°C for 10 minutes in 15 mL conical tubes. Cells were pelleted at 1350 x g at 4°C in a table top centrifuge and resuspended in 0.5 mL cold ChIP lysis buffer (50 mM HEPES-NaOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate) and sonicated to 200-1000 bp fragments using a VirSonic sonicator. Sonicated lysates were cleared by pelleting insoluble material at 13,000 RPM at 4°C followed by incubation with 5 ug antibody overnight. Next, Protein A magnetic beads (45 uL) were added to the lysate and incubated at 4°C for 7 hrs. Prior to addition, magnetic beads were washed 3 times with block (0.5% BSA/PBS). Immunoprecipitated material was washed 2 times each with ChIP lysis buffer, high salt lysis buffer (50 mM HEPES-NaOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate), and LiCl wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.5% sodium deoxycholate) and one time with TE plus NaCl, followed by elution and reverse crosslinking in 210 uL of 1% SDS in TE overnight at 65°C. 200 uL of uncrosslinked material was treated with RNase A for 2 hours, proteinase K for 2 hours, and extracted 2 times with phenol chloroform isoamyl alcohol. This was followed by ethanol precipitation with a glycogen coprecipitant, 80% ethanol wash and final resuspension in TE. Nucleic acid yield was determined via PicoGreen (Invitrogen). Adapter ligation and size selection (200-400 bp) were performed using a Beckman Coulter SPRI TE nucleic acid extractor, or, in some cases, performed by hand. Fragments were PCR amplified for 13 cycles followed by sequencing on an Illumina HiSeq 2000 system.
Adapter ligation and size selection (200-400 bp) were performed using a Beckman Coulter SPRI TE nucleic acid extractor, or, in some cases, performed by hand. Fragments were PCR amplified for 13 cycles followed by sequencing on an Illumina HiSeq 2000 system. For hand prepared libraries, libraries were made using the Ovation Library Prep kit according the manufacturer's instructions (NuGen). Briefly, DNA samples were end repaired, ligated to adaptors, and purified using AMPure beads. Purified samples were amplified by PCR for 18 cycles, purified, and sequenced on an Illumina HiSeq 2000 system.
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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 |
Reads were aligned using Bowtie.
Reads were extended 200 bp and grouped into 25-bp bins. Extended sequences were processed into Wiggle/BigWig format for visualization, allowing one repeat read.
Experimentally matched Input sequences were used to estimate background signal. A Poissonian model was used to determine statistically enriched bins with a p value threshold set at 1 X 10^-12 as described previously (Marson et al., 2008) and a 5-fold enrichment over input cutoff.
Genome_build: mm9
Supplementary_files_format_and_content: Bigwig values are read depth per 25 bp bin scaled per million mapped reads.
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| Submission date |
Mar 23, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jeffrey Michael Alexander |
| E-mail(s) |
jeffrey.alexander@ucsf.edu
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| Organization name |
Gladstone Institutes
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| Lab |
Bruneau Lab
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| Street address |
1650 Owens St
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94158 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE45447 |
Brg1 Modulates Enhancer Activation and Polycomb-mediated Repression in Mesoderm Differentiation [ChIP-Seq] |
| GSE45448 |
Brg1 Modulates Enhancer Activation and Polycomb-mediated Repression in Mesoderm Differentiation |
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| Relations |
| SRA |
SRX254812 |
| BioSample |
SAMN01990889 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1104462_Day4_H3K27me3_THF_Rep3.bw |
72.4 Mb |
(ftp)(http) |
BW |
| GSM1104462_Day4_H3K27me3_THF_Rep3_1e-12_enriched_regions.bed.gz |
63.1 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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