Instrument: Illumina HiSeq 2500
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: SINGLE
Construction protocol: E12.5 limb tissue was micordissected from wild type or HoxD mutants embryos and used for ChIP experiments. Samples were fixed in 1% formaldehyde/PBS for 10 (CTCF, H3K27ac, RAD21) or 25 (RAD21, SMC1) minutes at room temperature, washed three times with cold PBS containing protease inhibitor (and, for H3K27ac, deacetylase inhibitor) and stored at -80°C. For ChIP-seq samples, around, 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, protease inhibitor and, for the case of H3K27ac, deacetylase inhibitor) for 10 min on ice, and fragmented to a range of 200-500 bp using a Vibracell tip sonicator or a Diagenode Bioruptor pico waterbath device. DNA concentration was estimated using the Qubit ds DNA HS assay kit following manufacturer instruction and 25-30ug of chromatin were diluted ten times in ChIP dilution buffer (20mM HEPES, 150mM NaCl, 0.1% NP40) and incubated overnight at 4ºC with 4ug of anti-CTCF antibody (Active motif, 61311, 61312), anti-SMC1 (Bethyl, A300-055A), anti-RAD21 (abcam, ab992) or 2ug of anti-H3K27ac (abcam, ab4729) on a rotating platform. For ChIP-seq samples, the next day chromatin-antibody complexes were incubated with protein A/G agarose beads during 3h at 4ºC and successively washed following manufacturer instructions. Finally they were eluted and purified by phenol:chlorophorm extraction and precipitation. Between 5 to 10ng of immunoprecipitated DNA were sequenced with a 50bp single-end Illumina HiSeq flow cell. For ChIP-seq, 5-10 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.