|
| Status |
Public on Jun 07, 2013 |
| Title |
Andrey_FL_E10_d(8-13)d(a-S3)_K27m3 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Early forelimb buds E10.5
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6CBAF1/J
tissue: Forelimb buds
Stage: E10.5
genotype: del(attP-TpSB3)+/-/del(8-13)+/-
chip antibody: H3K27me3
chip antibody manufacturer: Millipore
chip antibody catalog #: 17-622
|
| Treatment protocol |
Early forelimb buds, presumptive digits and proximal segments of E12.5 forelimbs were micro-dissected from mouse embryos, fixed in 1% formaldehyde for 15 minutes at room temperature, washed three times with 4°C PBS and stored at -80°C. Pools of 40 early forelimbs (E9.5), 10 limb buds (10.5), 10 autopods or 10 Proximal forelimbs were used for each experiment. ChIP was performed according to (Lee et al., 2006), using 2 μg of anti-H3K4me3 (07-436, Millipore), H3K27me3 (17-622, Millipore) and EZview Red protein G/A Affinity Gel (Sigma). Immuno-precipitated and whole cell extract DNA (input) were de-crosslinked at 65°C overnight, treated with RNAseA, proteinase K and purified by phenol/chloroform extraction. ChIP and input DNA were amplified using ligation-mediated PCR (Lee et al., 2006), fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) and hybridized to custom tiling arrays (Affymetrix). For each sample, two independent ChIP-chip experiments were performed, with all antibodies.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Immuno-precipitated and whole cell extract DNA (input) were de-crosslinked at 65°C overnight, treated with RNAseA, proteinase K and purified by phenol/chloroform extraction.
|
| Label |
biotin
|
| Label protocol |
standard Affymetrix protocol
|
| |
|
| Channel 2 |
| Source name |
control
|
| Organism |
Mus musculus |
| Characteristics |
strain: B6CBAF1/J
tissue: Forelimb buds
Stage: E10.5
genotype: del(attP-TpSB3)+/-/del(8-13)+/-
chip antibody: input
|
| Treatment protocol |
Early forelimb buds, presumptive digits and proximal segments of E12.5 forelimbs were micro-dissected from mouse embryos, fixed in 1% formaldehyde for 15 minutes at room temperature, washed three times with 4°C PBS and stored at -80°C. Pools of 40 early forelimbs (E9.5), 10 limb buds (10.5), 10 autopods or 10 Proximal forelimbs were used for each experiment. ChIP was performed according to (Lee et al., 2006), using 2 μg of anti-H3K4me3 (07-436, Millipore), H3K27me3 (17-622, Millipore) and EZview Red protein G/A Affinity Gel (Sigma). Immuno-precipitated and whole cell extract DNA (input) were de-crosslinked at 65°C overnight, treated with RNAseA, proteinase K and purified by phenol/chloroform extraction. ChIP and input DNA were amplified using ligation-mediated PCR (Lee et al., 2006), fragmented and labeled using GeneChip WT Double-Stranded DNA Terminal Labeling Kit (Affymetrix) and hybridized to custom tiling arrays (Affymetrix). For each sample, two independent ChIP-chip experiments were performed, with all antibodies.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Immuno-precipitated and whole cell extract DNA (input) were de-crosslinked at 65°C overnight, treated with RNAseA, proteinase K and purified by phenol/chloroform extraction.
|
| Label |
biotin
|
| Label protocol |
standard Affymetrix protocol
|
| |
|
| |
| Hybridization protocol |
standard Affymetrix protocol
|
| Scan protocol |
standard Affymetrix protocol
|
| Description |
Chromatin immuno-precipitated with antibody against H3K27me3
|
| Data processing |
We used input gDNA for ChIP hybridisation to quantile normalized as in (Montavon et al., 2011). We scaled it to medial feature of 100 using TAS software (Affymetrix). For each genomic position, a data set was generated consisting of PM-MM pairs within a sliding window of 250 bp. The average ratios were plotted along the genomic DNA sequence in UCSC. Raw files are in mm8.
bedGraph files of normalized signals (ploted in mm9)
|
| |
|
| Submission date |
Mar 24, 2013 |
| Last update date |
Jun 07, 2013 |
| Contact name |
Marion LELEU |
| Organization name |
EPFL
|
| Department |
School of Life Sciences
|
| Lab |
Laboratory of developmental genomics
|
| Street address |
EPFL-SV-ISREC-UPDUB- Station 19
|
| City |
Lausanne |
| ZIP/Postal code |
1015 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL14183 |
| Series (2) |
| GSE45454 |
A Switch Between Topological Domains Underlies HoxD Genes Collinearity in Mouse Limbs (ChIP-chip) |
| GSE45457 |
A Switch Between Topological Domains Underlies HoxD Genes Collinearity in Mouse Limbs |
|
