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Sample GSM1135050 Query DataSets for GSM1135050
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
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
BioSample SAMN02141297
SRA SRX275809

Supplementary file Size Download File type/resource 230.5 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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