In situ Hi-C for mosquito embryos

Abstract The step-by-step Hi-C protocol is described for mosquito embryos at the 15-18 h developmental stage. In brief, we collect mosquito eggs, fix them with 3% formaldehyde, and prepare Hi-C libraries using nuclei isolated from ~1000-3000 embryos of mixed sexes. We use MboI restriction enzyme and T4-ligase to generate two biological replicates per species. Libraries can be prepared with NEBNext® Ultra™ II DNA Library Prep Kit for Illumina. The protocol was adapted for Anopheles species and successfully verified for Culex and Aedes embryos.