title: GSM2074596: E12.5 plus; Mus musculus; ChIP-Seq
library_construction_protocol: WT cortices (one litter of E12.5) were dissected, triturated in 1% formaldehyde in PBS and fixed for total of 10min at room temperature. Fixed cells were pelleted and washed with cold PBS. Pellets were lysed in 500ul of lysis buffer (1% SDS, 10mM EDTA and 50mM Tris, pH 8.1) on ice for 10min and lysates were sonicated using Bioruptor (Diagenode) on High Settings for 15 cycles (7.5 min of total sonication time). Resulting average chromatin size was 200-500 bp as verified by the Bioanalyzer. Cleared Chromatin was diluted 10 times with Dilution buffer (0.01% SDS, 1.1% Triton X- 100, 1.2mM EDTA, 16.7mM Tris-HCl, pH 8.1, 167mM NaCl) and was incubated overnight at 4C with 3ug of appropriate antibody. Mixture of protein A and G magnetic beads (Life Technologies) was preblocked overnight with BSA and tRNA and was added to the chromatin next day for 3 hours. Immune complexes were washed once with Low Salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 150mM NaCl), High Salt buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl, pH 8.1, 500mM NaCl), LiCl buffer (0.25M LiCl, 1% NP-40, 1% deoxycholic acid (sodium salt), 1mM EDTA, 10mM Tris, pH 8.1) and TE buffer. Complexes were eluted in 1% SDS, 10mM Sodium bicarbonate buffer at 65C for 10min. Crosslinks were reversed at 65C overnight, proteins were digested using proteinase K and DNA was purified using Zymo Chip DNA Clean and Concentrator kit (Zymo research). Immunoprecipitation was performed using the following antibody: Pbx1/2/3/ (sc-888, Santa Cruz). As a negative control in PBX ChIP experiments, the PBX antibody was incubated with PBX blocking peptide (sc-888P, Santa Cruz) at 1:400 molar ratio, and added to the chromatin lysates. ChIP-Seq libraries were prepared using NEBNext library Prep kit (New England Biolabs) and Illumina standard adaptors. 50bp single end.
attributes.tag: GEO Accession
attributes.value: GSM2074596