 |
 |
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Jul 06, 2014 |
Title |
ChIP-seq induced GFP-3Flag in neurospheres replicate 1 |
Sample type |
SRA |
|
|
Source name |
Neurospheres
|
Organism |
Mus musculus |
Characteristics |
strain: CD1 tissue: neurosphere treatment: Infection with 3XFlagIRESZsGFP chip antibody: anti-FLAG M2 (Sigma, F1804) chip antibody manufacturer: Sigma chip antibody catalog #: F1804
|
Treatment protocol |
Cortical progenitors were infected with 3XFlagFezf2IRESZsGFP or 3XFlagIRESZsGFP lentiviruses. 3XFlagFezf2IRESZsGFP or 3XFlagIRESZsGFP constructs were first cloned into pRETROX-IRES-ZsGreen1 retroviral vectors (Clontech Laboratories, Inc.) to generate high titer, replication-incompetent, VSVg-coated lentiviral particles that were packaged in 293T cells (MOI < 10^9 PFU/ml). 12-18 hours after plating, neural stem cells were infected with retroviruses. 16–20 h after infection, cells were switched into fresh media. Cells were collected 48 hours after infection for chromatin immunoprecipitation (ChIP).
|
Growth protocol |
Primary cortical progenitors were isolated from E14.5 brains and grown as neurospheres in presence of growth factors according to (Ahlenius et al., 2008). Neurospheres were expanded for a maximum of two passages and then plated as a monolayer on dishes coated with poly-D-lysine (VWR) and laminin (BD). Additional details on neurosphere isolation and culture are in Supplemental Experimental Procedures.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
ChIP analysis was performed on 15x10^7 neural stem cells per condition (3XFlagFezf2IRESZsGFP, 3XFlagIRESZsGFP and untransduced MOCK) as described (Nelson et al., 2006) with modifications as detailed in the Supplemental Experimental Procedures. Briefly, cells were dissociated with 0.25% trypsin (Invitrogen), washed with PBS and chemically cross-linked with formaldehyde solution (1%). Cells were disrupted in lysis buffer (50 mM Tris, pH 8.0; 10 mM EDTA; 1% SDS; and protease inhibitors) for 20 min on ice and sonicated with a Bioruptor to shear DNA into 200-700 bp fragments. Dynal magnetic beads (Sheep anti-mouse #M-28, Invitrogen, M2 FLAG) were preblocked with 5 μg of anti-FLAG M2 antibody per reaction (Sigma-Aldrich) overnight inverted at 4° C. Beads were then washed and resuspended in fresh 0.5% BSA diluted in PBS. Bead-antibody complexes were incubated at 4° C overnight with inversion. Beads were resuspended twice in low salt immune complex wash buffer (0.1% SDS; 1% Triton X-100; 2 mM EDTA; 20 mM Tris-HCl, pH 8.1; and 150 mM NaCl), incubated for 5 min at 4° C, with rotation, and resuspended in LiCl immune complex wash buffer (0.25 M LiCl; 1% NP40; 1% deoxycholate; 1 mM EDTA; and 10 mM Tris-HCl, pH 8.1) twice and incubated for 5 min at 4° C, with rotation. Beads were then rinsed with ice-cold TE and DNA was eluted in 100 μl of elution buffer at 65º for 15 minutes. Reverse crosslinking was performed by incubating the ChIP DNA overnight at 65º. DNA was purified on QIAquick PCR purification columns (Qiagen) for use as template for Solexa library construction. ChIP-seq libraries prepared using standard Illumina protocols
|
|
|
Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer II |
|
|
Description |
Chromatin IP against Flag
|
Data processing |
Basecalls performed using CASAVA version 1.4 Alignment: sequencing reads were aligned to the mouse genome (version mm9) using Bowtie version 0.12.7 with options "-q --best --strata -m 1 -p 4 --chunkmbs 1024", and only uniquely mapping reads were retained for further analysis. Peak-calling: GEM was used to detect binding events, using the options “--top 2000 --k_min 7 --k_max 12 --a 20 --q 3 --mrc 1 –constant_model_range --d_l 1000 --d_r 1000 --v 2 --nf --refine_pwm --gc0 0.42”. Reported peaks contain a ChIP-seq enrichment level that is significantly greater than 1.5 times the scaled read count from the corresponding region in the control experiment (p<10^-6, Binomial test, adjusted for multiple testing using Benjamini & Hochberg’s method). Genome_build: mm9 Supplementary_files_format_and_content: txt: GEM output files Supplementary_files_format_and_content: BED: Conversion of GEM output files to BED format, centered on the GEM binding event location Supplementary_files_format_and_content: BIGWIG: WIG files were generated using custom software to summarize read counts overlapping genomic regions. All reads were artificially extended out to 200bp in length. A bin size of 20bp was used for calculating overlapping read counts. WIG files were converted to bigWig using wigToBigWig (UCSC)
|
|
|
Submission date |
May 07, 2013 |
Last update date |
May 15, 2019 |
Contact name |
Shaun Mahony |
E-mail(s) |
mahony@psu.edu
|
Phone |
814-865-3008
|
Organization name |
Penn State University
|
Department |
Biochemistry & Molecular Biology
|
Lab |
Shaun Mahony
|
Street address |
404 South Frear Bldg
|
City |
University Park |
State/province |
PA |
ZIP/Postal code |
16802 |
Country |
USA |
|
|
Platform ID |
GPL9250 |
Series (1) |
GSE46707 |
Co-regulated gene expression downstream of a single transcription factor controls the acquisition of corticospinal motor neuron identity |
|
Relations |
BioSample |
SAMN02141297 |
SRA |
SRX275809 |
Supplementary file |
Size |
Download |
File type/resource |
GSM1135050_Arlotta_NS_48hr_G.control_GFP-3Flag_rep1.bw |
230.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
|
|
|
|
 |