Table 1:

Techniques used to study gene regulatory elements.

Technique	Purpose	Limitations
RNA-seq (29, 51) NGS of bulk RNA	Identify changes in transcriptional profile between different conditions	Doesn’t identify which DNA elements are enhancers. Is more hypothesis-generating than able to answer mechanistic questions.
ChIP-seq (Chromatin Immunoprecipitation) (8)(10) (44) Probe for histone modifications or specific transcription factors Follow ChIP with NGS ChIP-chip (ChIP followed by microarray) preceded this technique	Identify elements based on their histone modification or TF binding. See Figure 3 for typical signatures.	ChIP grade antibodies may not exist. Need to know specific protein or modification to target.
ATAC-seq (Assay for Transposase Accessible Chromatin) (15) Transposase targets areas of open chromatin that can be identified with NGS	Identify areas of accessible chromatin: areas likely to be functional regions actively involved in transcription	Does not identify function of specific regions, further studies needed to determine if promoter, enhancer, silencer etc.
Chromosome Conformation(40) (104) Cross link chromatin, digest, and ligate chromatin Depending on desired outcome, further manipulation followed by qPCR or NGS	Identify variety of interactions between one or many regulatory regions and their associated genes based on specific technology used 3C: interactions between a single pair of loci 4C: interactions between one loci and rest of the genome 5C: interactions between many loci of a particular region Hi-C and ChIA-PET: interactions genome-wide	Identifies two genomic regions interact but does not imply the element regulates a gene (i.e. a negative result can rule out enhancer activity, but a positive result does not confirm it is an enhancer). Resolution varies depending on specific technique. Some require specific knowledge of areas of interest (for primer development or for analysis).
STARR-seq (Self Transcribing Active Regulatory Region)(3) Insert putative regulatory sequence into reporter vector Perform RNA-seq to identify transcriptional effects of element CapStarr-seq(103) allows capture of specific target regions followed by enhancer activity assessment	Identify the effect of many different putative regulatory sequences on overall gene expression.	Integration into genome may not occur randomly, and bias towards stronger elements may occur. May not reflect in vivo genomic regulatory interactions. Suboptimal for genome-wide screen (CapStarr-seq circumvents this).
CAGE-seq (Cap Analysis of Gene Expression)(2) Reverse transcription allows capture of RNA-cDNA hybrids that can be processed into a library for NGS	Identify 5’ ends of nascent transcripts and determine transcriptional direction.	Detects only actively transcribed enhancers. Targets are labile.
CRISPR/Cas9-based CRISPR-based saturating mutagenesis (18) CRISPRi (39) CRISPR affinity purification in situ of regulatory elements (CAPTURE) (58)	Identify specific sequences necessary for enhancer function.	Possibility for confounding off-target effects. Suboptimal for genome-wide screen.

Teaching point: a wide variety of techniques utilizing next generation sequence have been developed that allow us to study the role enhancers play in gene regulation.