ChIP-seq Protocol for Embryonic Epidermal Tissue

Chromatin immunoprecipitation (ChIP) experiments were performed as previously described [64 (link)] with minor modifications. For each experiment, 50 posterior abdominal epidermes (A5, A6 and A7) of females between 0 and 2h after hatching and 3μg of antibody were used. Results present the mean of three independent experiments for each antibody. Tissue disruption was performed before cell lysis using the FastPrep technology (MP Biomedicals, Lysis matrix D, 20 seconds at 4m/s). Chromatin sonication was performed in a Bioruptor sonifier (Diagenode) (16 cycles of 30'' ON, 30'' OFF, High power). Input and immunoprecipitated DNA were purified with the Ipure kit (Diagenode) in 70μl of water and 4μl were used per qPCR reaction. qPCR experiments were carried out in a CFX96 system (Biorad) using SsoFast EvaGreen Supermix (Biorad). Primers used are listed in S1 Table. Data were normalized against input chromatin or panH3 ChIP. Antibodies used were anti-H3K4me3 (C15410003, Diagenode), anti-H3K27ac (C15410174, Diagenode), anti-panH3 (C15310135, Diagenode). Rabbit IgGs (Diagenode) were used as negative control (Mock).