Table 1.

Detailed description of in vitro methods that can be used to directly quantify the methylation sensitivity of TF binding.

Assay	Methodology	Advantages	Challenges	Ref
Methyl-HT-SELEX	• bead-based (automated) • random DNA ligand pool • 5mC probes assayed separately • ∼104 DNA reads per TF	• hundreds of parallel assays • universal probe design • allows for multiple rounds	• lower read count per TF due to high degree of multiplexing • risk of biased quantification due to separate ligand pools	[45]
EpiSELEX-seq	• EMSA-based (manual) • random DNA ligand pool • 5mC probes barcoded and assayed in pooled sample • input DNA and bound DNA sequenced separately • ∼106 DNA reads per TF	• accurate quantification of relative enrichment • full sequence spectrum covered	• single TF (complex) assayed • limited to single round of enrichment	[44]
Methyl-Spec-seq	• EMSA-based (manual) • random DNA ligand pool • 5mC probes barcoded and assayed in pooled sample • bound and unbound DNA sequenced separately • ∼106 DNA reads per TF	• EMSA-based (manual) • dedicated probe design	• single TF (complex) assayed • limited to single round of enrichment	[51]
Methyl-PBM	• DNA microarrays • fluorescently labeled TF • ∼105 dsDNA probes	• no DNA sequencing needed	• DNA microarray needed	[39, 46, 50]
(amp)DAP-seq	• fragments of genomic DNA • recombinant TF proteins	• natural epigenetic context	• quantification challenging • methylome data required	[52, 53]
Classic low-throughput methods	isothermal calorimetry (ITC) or electromobility shift assay (EMSA) or DNase footprinting	accurate quantification (Kd) and flexible sequence design	low throughput