A front limb of a quadruped. (The Random House College Dictionary, 1980)
Attributes by original data submitter
Sample
source_name
embryonic forelmb bud, HoxD13 ChIP
strain
Mixed C57BL/6 - CD1
developmental stage
gestational day e11.5
tissue
forelimb bud
antibody
anti-HoxD13 (in-house, affinity purified)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Chromatin immunoprecipitation were performed as previously described (PMID: 16630818) with some modifications. Briefly, E11.5 ZAP cells from wild type and mutant limbs were cross-linked with 1% formaldehyde at room temperature for 10 min. Chromatin was cross-linked using formaldehyde as previously described except for HOXA13 ChIP for which we performed the cross-linking using a dual method using disuccinimidyl glutarate (DSG) and formaldehyde (PMID: 21123453). Chromatin was sonicated to obtain fragments with an average size ranging between 100-600 bp. Protein A and G Dynabeads (Invitrogen) were incubated 6h at 4 C with 5 µg of antibody. The chromatin was then incubated with the beads overnight at 4 C. Immuno-complexes were sequentially washed. The protein/DNA complexes were eluted in an SDS buffer (1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA) at 65 C overnight. Samples were treated for 15 min RT with RNase A and then with Proteinase K for one hour at 55 C. Finally, the DNA was purified on QIAquick columns (Qiagen) and the quality was assessed on the Agilent bioanalyzer. The ChIP-seq libraries were prepared by the IRCM functional genomics core facility using Illumina TruSeq library preparation kit followed by sequencing on an Illumina HiSeq 2000.