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SRX091255: Negative control for ChIP-SEQ of ETV3-HA in 293T cells (cells not transfected with ETV3-HA)
1 ILLUMINA (Illumina Genome Analyzer II) run: 28.9M spots, 1G bases, 602.7Mb downloads

Design: Three 15-cm plates of HEK 293T cells were transfected (or not for negative control) with HA-tagged human ETV3 (20 µg plasmid, 60 µL FuGene HD in 0.5 mL PBS) for two days to a density of 25e6 cells per plate. Cells were treated for 20 minutes with U0126 (10 µM) and/or PMA (100 nM). A control experiment was conducted with non-transfected cells. Frozen pellets of formaldehyde-fixed cells were resuspended and lysed. The cells were then sonicated using a Misonix Sonicator 3000 at 33W for twelve cycles of a 20 second pulse followed by a 60 second pause. Lysate was centrifuged at 14,000 rpm for 10 minutes at 4°C to remove impurities. The lysate was incubated with 50 µL of Dynal Protein G magnetic beads bound to 10 µg of antibody for 15 hours at 4°C. The purified DNA was prepared for sequencing on a Beckman Coulter SPRI-TE following manufacturer’s instructions. The seq-prepped DNA was PCR amplified using Illumina primers for 18 cycles. Samples were sequenced using Illumina Solexa Genome Analyzers 2.0. Reads were mapped to the hg18 reference genome using the GERALD alignment software package. Uniquely mapping reads were run through the MACS v1.4.0beta peak calling software using default parameters.
Submitted by: (MIT_BL)
Study: Large scale discovery of ERK2 substrates identifies transcriptional regulation by ETV3
show Abstracthide Abstract
The mitogen-activated protein kinase ERK2 is ubiquitously expressed in mammalian tissues and is involved in a wide range of biological processes. Although MAP kinases are heavily studied, identification of their substrates remains challenging. We have optimized a chemical genetic system using an analog sensitive kinase to tag and isolate ERK2 substrates in vitro. This approach identified 80 proteins phosphorylated by ERK2, 13 of which are previously reported, across a wide range of pathways including regulation of transcription and translation, Rho GTPase activity and mRNA processing. Among the novel substrates, the ETS repressor factor ETV3 is heavily phosphorylated on sites within canonical and non-canonical ERK motifs. Phosphorylation of ETV3 regulates transcription by preventing binding to DNA at promoters for several thousand genes, including targets involved in negative feedback regulation of itself and of upstream signals.
Sample: 293T ETV3-HA ChIP-SEQ negative control (no HA-ETV3)
SAMN00708945 • SRS256991 • All experiments • All runs
Organism: Homo sapiens
Library:
Name: ETV3-HA negative control
Instrument: Illumina Genome Analyzer II
Strategy: ChIP-Seq
Source: GENOMIC
Selection: size fractionation
Layout: SINGLE
Spot descriptor:
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Pipeline: show...hide...
ProgramVersion
GERALD
MACS1.4.0 beta
Runs: 1 run, 28.9M spots, 1G bases, 602.7Mb
Run# of Spots# of BasesSizePublished
SRR33010828,859,9451G602.7Mb2011-09-26

ID:
104050

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