Nucleolus and centromere Tyramide Signal Amplification-Seq reveals variable localization of heterochromatin in different cell types.

Genome differential positioning within interphase nuclei remains poorly explored. We extended and validated TSA-seq to map genomic regions near nucleoli and pericentric heterochromatin in four human cell lines. Our study confirmed that smaller chromosomes localize closer to nucleoli but further deconvolved this by revealing a preference for chromosome arms below 36-46 Mbp in length. We identified two lamina associated domain subsets through their differential nuclear lamina versus nucleolar positioning in different cell lines which showed distinctive patterns of DNA replication timing and gene expression across all cell lines. Unexpectedly, active, nuclear speckle-associated genomic regions were found near typically repressive nuclear compartments, which is attributable to the close proximity of nuclear speckles and nucleoli in some cell types, and association of centromeres with nuclear speckles in hESCs. Our study points to a more complex and variable nuclear genome organization than suggested by current models, as revealed by our TSA-seq methodology. Nucleolar and PCH TSA-Seq to map the mean cytological proximity of chromosomes genome-wide relative to nucleoli and pericentric heterochromatin (PCH) in four human cell lines - K562, HFFc6, HCT116, H1-hESC. Different condition were used: 1) Condition C: labeling with 1:3000 tyramide biotin, 50% sucrose and 0.0015% hydrogen peroxide, 2) Condition E: labeling with 1:300 tyramide biotin, 50% sucrose and 0.0015% hydrogen peroxide (minor modification: 150ul of Dynabeads M-270 streptavidin (Invitrogen, catalog no. 65306) was used to purify the biotinylated DNA) 3) Condition AI for HFFc6, H1, and K562 and 4) Condition A for HCT116 cells.