GSM2535578: DFL_E12_WT_CTCF; Mus musculus; ChIP-Seq
Micro-dissected distal segments of E12.5 limbs from wild type CD1 mice, were used for ChIP-seq. Samples were fixed in 1% formaldehyde/PBS for 10 minutes at room temperature, washed three times with cold PBS containing protease inhibitor and stored at -80°C.  One hundred milligrams of tissue  were used. Nuclei were extracted by incubating them in a cell lysis buffer provided in the ChIP IT high sensitivity kit (Active motif) during at least 10 minutes and with the aid of A Dounce homogenizer type B (Active motif). The isolated nuclei were pelleted and lysed in sonication buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH=8.0 and protease inhibitor) for 10 min on ice, and fragmented to a range of 200-500 bp using a Vibracell tip sonicator. DNA concentration was estimated using the Qubit ds DNA HS assay kit following manufacturer instruction and 25 ugr of chromatin were diluted ten times in ChIP dilution buffer (20mM HEPES, 150mM NaCl, 0.1% NP40) and incubated overnight at 4C with 4mgr of anti-CTCF antibody (Active motif) on a rotating platform. The next day chromatin-antibody complexes were incubated with protein A/G agarose beads during 3h at 4C and successively  washed following manufacturer instructions. Finally they were eluted and purified by phenol:chlorophorm extraction and precipitation. A total of 10ngr of immunoprecipitated DNA were sequenced with a 50bp single-end Illumina HiSeq flow cell. For ChIP-seq, 10 ng of purified DNA were used to make libraries according to the manufacturer’s protocol (Illumina). The material was sequenced with 50 bp single-end reads on the Illumine HiSeq according to the manufacturer’s specifications.