TSA-Seq Mapping of Nuclear Genome Organization [seq]

We describe TSA-Seq, a new mapping method able to estimate mean chromosomal distances from nuclear speckles genome-wide and predict several Mbp chromosome trajectories between nuclear compartments without sophisticated computational modeling. Ensemble-averaged results reveal a clear nuclear lamina to speckle axis correlated with a striking spatial gradient in genome activity. This gradient represents a convolution of multiple, spatially separated nuclear domains, including two types of transcription ?hot-zones?. Transcription hot-zones protruding furthest into the nuclear interior and positioning deterministically very close to nuclear speckles have higher numbers of total genes, the most highly expressed genes, housekeeping genes, genes with low transcriptional pausing, and super-enhancers. Overall design: We used TSA-Seq to study genome organization relative to nuclear speckles and the nuclear lamina. For nuclear speckles, we performed TSA-Seq using two different antibodies against the protein SON and an antibody against a phosphorylated epitope of the protein SC35. We also performed TSA-Seq using an antibody against RNA Pol II as a general marker for active nuclear domains to compare with the speckle TSA-Seq chromosome patterns. For the nuclear lamina, we performed TSA-Seq using antibodies against lamin B and Lamin A/C. For each TSA-Seq experiment, a no primary control experiment was done in parallel, where the same TSA-Seq procedure is performed but with no added primary antibody.