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Sample GSM1175111

Query DataSets for GSM1175111

Status

Public on Aug 13, 2013

Title

Background_Basal

Sample type

SRA

Source name

Neural progenitor cells

Organism

Mus musculus

Characteristics

strain: FVB/N
cell type: Primary adult neural progenitor cell
passages: 4-9
chip antibody: No Antibody

Treatment protocol

To induce FOXO3 nuclear accumulation, cells were incubated in low growth factor signaling conditions: Neurobasal A medium supplemented with penicillin/streptomycin/glutamine and 2% B27 for 4 hours, followed by a 1.5 hour incubation with 20 mM LY294002 (LY, Calbiochem-Novabiochem Corp.), a specific PI3K inhibitor, to inhibit residual growth factor signaling.

Growth protocol

Adult (12-week-old) mouse NPCs were isolated as previously described (Renault et al., Cell Stem Cell 2009). Briefly, whole mouse forebrains were homogenized and incubated for 30 min in HBSS (Invitrogen) with 1 U/ml DispaseII (Roche), 250 U/ml DNaseI (Sigma) and 2.5 U/ml Papain (Worthington) at 37oC. After mechanical dissociation, cells were purified by sequential 25% and 65% Percoll (Amersham) gradients. Cells were cultured in high growth factor signaling conditions: Neurobasal A (Invitrogen) medium supplemented with penicillin/streptomycin/glutamine (Invitrogen), 2% B27 (Invitrogen) and 20 ng/ml each of FGF2 (Peprotec) and EGF (Peprotec).

Extracted molecule

genomic DNA

Extraction protocol

Chromatin was cross-linked with 1% formaldehyde for 10 min, followed by quenching with 0.125 M glycine for 5 min. Cells were washed and incubated in swelling buffer (10 mM HEPES pH 7.8, 1.5 mM MgCl2, 10 mM KCl, 0.1% NP-40, 1 mM DTT, 0.5 mM PMSF) for 15 min on ice, followed by nuclei isolation by dounce homogenization. Nuclei were pelleted and resuspended in RIPA buffer (1% NP-40, 0.5% sodium deoxycholate, 1% SDS in PBS pH 7.4), and chromatin was sheared with a Vibra-Cell Sonicator VC130 (Sonics) six times for 30 sec at 60% amplitude.
Single end libraries were generated according to the manufacturer’s instructions (Illumina).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer IIx

Data processing

Reads with low quality scores (sequences that have phred <15 on 85% seq) were filtered using the fastx toolbox (fastq_quality_filter -v -Q 33 -q 15 -p 85)
Reads were aligned to mm9 using bowtie-0.12.7 (bowtie -p 4 -S -q -v 2 -m 3 --best --strata)
For chromatin marks, peaks were generated using Macs (version 2) software with "broad option" and with default settings (macs2 --broad)
For transcription factor, peaks were generated using QuEST 2.1 using the highest stringency peak calling parameters (Fold enrichment > 50, bandwidth=30)
Genome_build: mm9
Supplementary_files_format_and_content: H3K27me3, H3K4me1 and H3K4me1 peak files were generated using MACS 2, Ascl1 and FoxO3 peak bed files were generated using QuEST 2.1

Submission date

Jun 26, 2013

Last update date

May 15, 2019

Contact name

Anne Brunet

Organization name

Stanford University

Department

Genetics

Street address

300 Pasteur Drive,