Figure 3

Next-generation sequencing (NGS) techniques to identify gene regulatory elements. For RNA-Seq, complementary DNA (cDNA) is generated from RNA of interest, fragmented either as cDNA or RNA, followed by the ligation of sequencing adapters. In chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq), chromatin is crosslinked with formaldehyde, fragmented and then immunoprecipitated with a specific antibody. The crosslinking is then removed and sequencing adapters are ligated. For next-generation sequencing of deoxyribonuclease I (DNaseI) hypersensitive sites (DNase-Seq), chromatin is digested with DNase I. One biotinylated adapter is ligated and then the fragments are digested with restriction enzyme MmeI and subjected to biotin pull-down following which a second adapter is ligated. In formaldehyde-assisted isolation of regulatory elements (FAIRE)-Seq, chromatin is crosslinked with formaldehyde and then fragmented via sonication. Fragments are subjected to phenol-chloroform extraction and sequencing adapters are ligated to fragments recovered in aqueous phase. For chromatin interaction analysis by paired-end tag sequencing (ChIA-PET), chromatin is crosslinked with formaldehyde and then fragmented via sonication. An antibody is used to enrich for protein-bound fragments. Biotinylated half-linkers with MmeI sites are ligated. Half-linkers are connected, and linked fragments are digested with MmeI and selected for by biotin pull-down. Sequencing adapters are then ligated. In high-throughput chromosome capture (Hi-C), chromatin is crosslinked with formaldehyde, fragmented using a restriction enzyme and ends are labeled with biotin and ligated. DNA fragments are then sheared and biotin-containing fragments are enriched via biotin pull-down. Sequencing adapters are then ligated.