Table 1:

Overview of Tools and Techniques for the Study of Promoter Proximal Pausing

Type	Tool/Technique	Utility
Genome-wide Pol II and transcription factor mapping	ChIP, ChIP-seq, ChIP-exo, ChIP-nexus	Determination of the location of Pol II and its binding partners
Genome-wide Pol II mapping	Permanganate ChIP-seq, GRO-seq, PRO-seq, ChRO-seq, NET-seq, chromatin-associated nascent transcript sequencing	Mapping of transcriptionally engaged Pol II
Pol II mapping	Nuclear walk-on assay	Quantitative assessment of pausing
Pol II mapping	Reporter ChIP-nexus	Evaluation of Pol II pausing on transfected plasmids
Genome-wide Pol II and transcription factor mapping	Drosophila polytene chromosome staining	Observing association of Pol II and other factors such as NELF and DSIF with chromosomes
Cell free	Nuclear extract pausing assays	Study of individual factors in semi in vivo conditions
Cell free	Electrophoretic mobility shift assay	Examination of binding effectiveness between Pol II and pausing factors; useful for targeted mutagenesis studies of NELF and DSIF domains
Cell free	Reconstituted pausing assay using purified Pol II, DSIF, NELF. HSF1, and PIC components	Potentially transformative for in vitro mutagenesis-function studies of pausing factors
Drug	Triptolide	Inhibition of Pol II initiation; useful for estimates of Pol II pause half life
Drug	Flavopiridol, DRB, other P-TEFb inhibitors	Inhibition of Pol II pause release
Drug	Pol II-DSIF inhibitors	May provide significant insight into the role of DSIF in pausing
Structural data	Structures of Pol II-DSIF-NELF and Pol II-DSIF-Spt6-PAF	Critical roadmaps for designing biochemical studies