A front limb of a quadruped. (The Random House College Dictionary, 1980)
Attributes by original data submitter
Sample
source_name
forelimb bud
strain background
Swiss Webster
tissue/chip rxn
~400,000 cells/ChIP
age
E10.5 (32-35 somites)
chip antibody
H3K4me2 (millipore 07-030)
genotype or treatment
Shh-/-
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were fixed for 15 minutes at room temperature with 1% formaldehyde then lysed. After cell lysis, chromatin was sheared in shearing buffer containing 0.25% SDS using a Covaris S2 focused ultrasonicator. Sheared chromatin was incubated with antibody coated dynabead preparations (1ug antibody with 20ul beads) overnight. Beads were washed 5 times with RIPA buffer (1% NP40, 0.7% Sodium Deoxycholate, 1mM EDTA pH8, 50mM Hepes-KOH pH7.5, 2% w/v Lithium Chloride) and then eluted at 70°C for 15 minutes in elution buffer (50mM TRIS pH 8, 10mM EDTA pH 8, 1% SDS). Crosslinks were reversed overnight at 70°C and chromatin was purified and concentrated by phenol chloroform extraction and EtOH precipitation. One third of each ChIP sample was used for library preparation. Library preparation was done according to the Broad Pond method, (PMCID: PMC3091298) using the NEBNext library preparation kit (NEB E6040L) using non-standard 15 cycles of PCR amplification.