|
| Status |
Public on Dec 22, 2011 |
| Title |
Whole_Forebrain_2 [ChIP-chip 244K array, 2 of 2] |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Embryonic brains [IP DNA]
|
| Organism |
Mus musculus |
| Characteristics |
strain: Swiss outbred (RJOrl:SWISS)
tissue: brain
developmental stage: E15.5
chip antibody: anti-Arx antibody (ARX C-14)
chip antibody vendor: Santa-Cruz
chip antibody lot#: A2108
chip antibody catalog#: sc-48843
|
| Treatment protocol |
CELLS : Cells were transfected with an expression vector expressing Arx using Lipofectamine 2000 according to the guideline of the manufacturer (Invitrogen). Two days after transfection, cells were fixed in 1% formaldéhyde, followed by the addition of glycine. BRAINS : Embryonic brains disaggregation and homogenization were performed in liquid nitrogen using a pestle and a mortar. Samples were fixed in a solution containing PBS, formaldehyde and protease inhibitor cocktail. Reaction was stopped by the addition of glycine.
|
| Growth protocol |
CELLS : N2a cells were grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (PAA) which was supplemented with 10% fetal bovine serum gold (PAA), 2 mM L-glutamine (Lonza), 0,1 mM non essential amino-acids (Gibco) and 2 mM penicillin/streptomycin (PAA). Cells were grown at 37°C with 5% CO2. BRAINS : Swiss outbred (RJOrl:SWISS) mice (Janvier, France) were used in all the studies. Mouse brains were extracted from day 15,5 embryos in cold PBS. Following brain isolation, the whole forebrain (ventral telecenphalon, thalamus, cerebral cortex and olfactory bulbs) were frozen in liquid nitrogen.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA of cells and embryonic brains was fragmented by sonication. Immunoprecipitation was perfomed using anti-Arx antibody (ARX C-14; Santa-Cruz; Catalog sc-48843; Lot A2108). Immune complexes were captured by addition of dynabeads protein G. Following washes with gradient stringent buffers, protein-DNA complexes were eluted. Reverse cross-link was performed then, immunoprecipitated DNA and Input DNA were isolated via phenol-chloroform extraction followed by ethanol precipitation.
|
| Label |
Alexa Fluor 647
|
| Label protocol |
Immunoprecipitated chromatin and total Input DNA were amplified using GenomePlex Complete Whole Genome Amplification kit (Sigma).Then, two micrograms of IP and Input DNA were labelled with Alexa Fluor 647 or Alexa Fluor 555 respectively, by use of the BioPrime Plus Array CGH Indirect Genomic Labbeling System (Invitrogen).
|
| |
|
| Channel 2 |
| Source name |
Embryonic brains [Input DNA]
|
| Organism |
Mus musculus |
| Characteristics |
strain: Swiss outbred (RJOrl:SWISS)
tissue: brain
developmental stage: E15.5
|
| Treatment protocol |
CELLS : Cells were transfected with an expression vector expressing Arx using Lipofectamine 2000 according to the guideline of the manufacturer (Invitrogen). Two days after transfection, cells were fixed in 1% formaldéhyde, followed by the addition of glycine. BRAINS : Embryonic brains disaggregation and homogenization were performed in liquid nitrogen using a pestle and a mortar. Samples were fixed in a solution containing PBS, formaldehyde and protease inhibitor cocktail. Reaction was stopped by the addition of glycine.
|
| Growth protocol |
CELLS : N2a cells were grown in Dulbecco's Modified Eagle Medium (DMEM) high glucose (PAA) which was supplemented with 10% fetal bovine serum gold (PAA), 2 mM L-glutamine (Lonza), 0,1 mM non essential amino-acids (Gibco) and 2 mM penicillin/streptomycin (PAA). Cells were grown at 37°C with 5% CO2. BRAINS : Swiss outbred (RJOrl:SWISS) mice (Janvier, France) were used in all the studies. Mouse brains were extracted from day 15,5 embryos in cold PBS. Following brain isolation, the whole forebrain (ventral telecenphalon, thalamus, cerebral cortex and olfactory bulbs) were frozen in liquid nitrogen.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA of cells and embryonic brains was fragmented by sonication. Immunoprecipitation was perfomed using anti-Arx antibody (ARX C-14; Santa-Cruz; Catalog sc-48843; Lot A2108). Immune complexes were captured by addition of dynabeads protein G. Following washes with gradient stringent buffers, protein-DNA complexes were eluted. Reverse cross-link was performed then, immunoprecipitated DNA and Input DNA were isolated via phenol-chloroform extraction followed by ethanol precipitation.
|
| Label |
Alexa Fluor 555
|
| Label protocol |
Immunoprecipitated chromatin and total Input DNA were amplified using GenomePlex Complete Whole Genome Amplification kit (Sigma).Then, two micrograms of IP and Input DNA were labelled with Alexa Fluor 647 or Alexa Fluor 555 respectively, by use of the BioPrime Plus Array CGH Indirect Genomic Labbeling System (Invitrogen).
|
| |
|
| |
| Hybridization protocol |
5 µg of labelled DNA was hybridized to mouse promoter 244K ChIP-Chip microarrays set (Agilent Technologies) for 40 hours, at 65°C, at 20 rpm.
|
| Scan protocol |
Hybridization images were obtained using an Agilent DNA microarray scanner (G2505B). Scan Region : Agilent HD (61 x 21,6 mm) / Resolution : 5 µm / TIFF : 16 bit / Red PMT : 100% / Green PMT : 100%. Intensity data were extracted using Feature Extraction software (FE v9,5,1) (Agilent Technologies).
|
| Description |
ChIP_ARX5_12233
|
| Data processing |
Intensity data on each array were normalized with the Lowess method. To identify regions of significant Arx association, the enrichment of each probe on the array was calculated as the ratio of the intensities of Arx-immunoprcipitated DNA (y-axis) to control Input chromatin (x-axis). One first important assumption is that points corresponding to non positive probes are distributed symmetrically around the axis x=y. A threshold spline curve was then defined by considering only the probes below this axis. Reporting this curve above the axis allowed discrimination between positive and negative probes. For each probe, a P-Value was obtained using a normalised distance between it and the axis x=y.
|
| |
|
| Submission date |
Jun 15, 2011 |
| Last update date |
Dec 22, 2011 |
| Contact name |
Gaelle Friocourt |
| E-mail(s) |
[email protected]
|
| Phone |
+33 298443895
|
| Fax |
+33 298467910
|
| Organization name |
Inserm U613
|
| Department |
Molecular Genetics
|
| Street address |
46 rue Félix Le Dantec, CS51819
|
| City |
Brest |
| ZIP/Postal code |
29218 |
| Country |
France |
| |
|
| Platform ID |
GPL4129 |
| Series (2) |
| GSE29985 |
Identification by ChIP-on-Chip of ARX target genes, a transcription factor implicated in mental retardation and epilepsy |
| GSE30191 |
ARX transcription factor implicated in mental retardation and epilepsy |
|
