A front limb of a quadruped. (The Random House College Dictionary, 1980)
Attributes by original data submitter
Sample
source_name
distal forelimb
developmental stage
E12.5
chip-antibody
PolII (Abcam, ab5131, lot GR37362-1)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Distal forelimbs were collected in 1X PBS containing 10% FBS and the samples were incubated with collagenase to obtain single-cell suspensions The cells were then fixed with 1% formaldehyde at room temperature. Crosslinking was stopped with glycine. Cell pellets were flash frozen on dry ice. Cells were sonicated to obtain fragments with average sizes ranging between 100-600 bp. Protein G Dynal Beads (Invitrogen) were incubated 8 hours at 4°C with antibodies. The chromatin was then incubated with the beads overnight. Immunoprecipitated 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). Crosslinks were reversed. Samples were treated with RNAseA and then with Proteinase K. Finally, the DNA was purified on QIAquick columns (Qiagen). Libraries were prepared according to Illumina's instructions