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Sample GSM4546393

Query DataSets for GSM4546393

Status

Public on Nov 26, 2020

Title

ChIP-Seq_PAX6_14

Sample type

SRA

Source name

Enteric precursor cells

Organism

Mus musculus

Characteristics

strain: CD1
cell type: Enteric precursor cells
age: P7
tissue: ganglionic gut
chip antibody: Anti-PAX6 Antibody (Millipore, USA)
pool number: Pool3

Growth protocol

Enteric precursor cells were extracted from ganglionic bowels of 7 days aged CD-1 mice. Bowels were dissected and incubated in a solution of 0.26 mg/ml trypsin collagenase, 5 mg/ ml dispase, 0.28 mg/ml hyaluronidase, 3.3 μg/ml elastase, and 0.6 mg/ml collagenase in phosphate-buffered saline for up to 30 minutes at 37 °C. Digested tissue was triturated and washed, and the cells were cultured in Dulbecco’s modified Eagle medium (1 mg/ml glucose) containing 100 U/ml penicillin, 100 g/ml streptomycin, supplemented with 2 mmol/l l-glutamine (Gibco Life Technologies, Carlsbad, CA), 0.05 mmol/l 2-mercaptoethanol, 1% (volume/volume) N1 (Sigma Aldrich, Poole, Dorset, UK), 10% (volume/volume) human serum, 20 ng/ml basic fibroblast growth factor, 20 ng/ml epidermal growth factor, and 10 ng/ml glial cell–derived neurotrophic factor (Peprotech, London, UK).

Extracted molecule

genomic DNA

Extraction protocol

Cells were fixed with 1% formaldehyde and was sonicated in a Bioruptor System (Diagenode, USA). ChIP assay was performed with the Magna Chip Hisens Kit (Millipore,USA) according to manufacturer’s instructions and using the Anti-Pax6 antibody (Millipore, USA). 10% of samples volume/reaction was kept as controls (input) before immunoprecipitation. Mouse IgG (Diagenode) was used as negative control (IgG).
ChIP, input and IGG samples were processed with the ChIP-Seq DNA sample prep kit (Illumina, USA), following the manufacturer’s instructions. Size-selection was then performed by agarose gel electrophoresis to isolate fragments with size between 200 and 300bp. Afterwards, purified libraries were amplified by PCR and size range was subsequently checked in the Agilent 2100 Bioanalyzer using the High Sensitivity DNA Kit (Agilent, USA). Quantification was performed in the 7500-Real Time PCR System (Applied Biosystems) with the Library Quantification Kit (Kapa Biosystems). After proper dilutions, 4 nM libraries were processed, to obtain pools at a final concentration of 8 pM following the instructions of the Miseq Reagent kit V2. (50 cycles) (Illumina, USA).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina MiSeq

Data processing

Basecalls performed automatically by MiSeq Control Software using Real-Time Analysis
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie for Illumina (Galaxy Version 1.1.2) with default parameters
Peaks were called using MACS Model-based Analysis for ChIP-Seq (version 1.0.0) with default parameters
BETA-minus: Targets prediction with binding only (version 1.0.0), PAVIS and PAPST 1.0 tools, were used to assign the ChIP-seq peak data to the neighboring genes (annotation) with default parameters
Genome_build: mm9
Supplementary_files_format_and_content: Bed files were generated using MACS Model-based Analysis for ChIP-Seq (version 1.0.0); Columns: Chr peak; Start peak; End peak; -10*log10(pvalue)

Submission date

May 11, 2020

Last update date

Nov 26, 2020

Contact name

Villalba Leticia

E-mail(s)

leticia.villalba.benito@hotmail.es

Organization name

FISEVI

Street address

Avda. Manuel Siurot s/n