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| Status |
Public on Mar 01, 2015 |
| Title |
Day4_Brg1_ChIPexo_4OHT_Rep1 |
| 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
agent: 4OHT
timepoint: Day 4
chip antibody: anti-BRG1 (Abcam, catalog# 110641, lot#YG081901C)
brg1 levels: Deleted
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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 |
Frozen pellets of cross-linked cells (8-10x106) 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 G magnetic beads (45 uL) were added to the lysate and incubated at 4°C for 7 hrs.
ChIPExo was performed on Brg1 ChIP material while still on protein G magnetic dynabeads. After incubation with the antibody, the beads were washed six times with RIPA buffer (50mM HEPES, pH7.6, 1mM EDTA, pH8.0, 0.7% Sodium deoxycholate, 1% NP-40 and 500mM Lithium chloride) and two times with Tris (10mM Tris.Cl, pH 8.0). The bead bound DNA were end polished at 30°C for 30mins in using T4 DNA polymerase, Klenow fragment of DNA polymerase and T4 polynucleotide kinase. From this point each subsequent enzymatic steps were followed by two washes with each of RIPA and 10mM Tris. P7 adapter was ligated to the DNA ends using T4 DNA ligase at 25°C for 60 mins followed by nick repair using Phi29 polymerase at 30°C for 20 mins. Samples were periodically vortexed at 900rpm in a thermomixure during enzymatic reaction. DNA was digested with lambda and RecJf exonucleases at 37°C for 30 mins each. Samples were then eluted off the beads with 100ml of Elution buffer (50mM Tris.Cl, pH 8.0, 10mM EDTA, 1% SDS) by incubating at 65°C for 30mins with periodic shaking. RNase A was added to the samples for 30min at 37°C. Proteinase K was added to degrade proteins, crosslink was reversed by overnight incubation at 65°C and using Ampure beads ChIP DNA was purified. The purified DNA was denatured at 95°C for 5 mins before synthesis of the 2nd strand by P7 primer extension in presence of Phi29 polymerase. Then, P5 adapters were ligated to the DNA ends and the DNA fragments were PCR amplified for 18 cycles using universal primers containing the index sequences. PCR products were purified using Ampure beads, size selected using 2% agarose gels in an E-Gel Electrophoresis system (Invitrogen), gel purified using minielute gel extraction columns (Qiagen) and eluted in 20microlitre of TE. Samples were quantified and analysed on Qubit and Bioanalyser before sequencing on a Illumina Hi-Seq 2500 sequencing machine.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
ChIP-exo-enriched genomic DNA
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| Data processing |
The 50bp single-end reads were trimmed for quality using fastq-mcf (Aronesty et al 2011), and were them aligned to the mm9 genome assembly using Bowtie2 (Langmead et al 2012).
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.
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 |
Dec 09, 2014 |
| 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 |
GPL17021 |
| Series (2) |
| GSE45448 |
Brg1 Modulates Enhancer Activation and Polycomb-mediated Repression in Mesoderm Differentiation |
| GSE63976 |
Brg1 Modulates Enhancer Activation and Polycomb-mediated Repression in Mesoderm Differentiation [ChIP-exo] |
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| Relations |
| BioSample |
SAMN03256254 |
| SRA |
SRX800824 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1561594_Day4_Brg1_ChIPexo_4OHT_Rep1.bw |
93.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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