Extended Data Fig. 3: Additional information on the single-molecule Pol II–nucleosome transcription assays.
From: Phase-separated NDF−FACT condensates facilitate transcription elongation on chromatin
a, Median residence time (seconds per bp) histograms of Pol II transcription through a nucleosome in the absence (red, n = 30 biological replicates), or presence of NDF (green, n = 24 biological replicates), FACT (cyan, n = 27 biological replicates), or both FACT and NDF proteins (yellow, n = 25 biological replicates). b, Schematic of the tweezing chamber used for the C-trap (Lumicks) experiments. Representative trace of a single Pol II transcribing through the molecular ruler and nucleosome, in the presence of both (c) or either (e) NDF or (f) FACT alone, accompanied by the corresponding fluorescence kymographs. Position of Cy5 molecule is shown (white arrow). The entry, dyad and exit of the nucleosome are indicated by black dashed lines. d, Fluorescence intensity profile of the confocal image depicted in Fig. 3e. The total number of photon counts for each laser was determined by delineating the region of interest, that is, where the condensates interact with the Pol II–nucleosome transcription system (yellow box). g-h, Relationship between NDF-FACT condensate size and Pol II nucleosome transcriptional efficiency. (g) Scatter plot showing nucleosome crossing time as a function of condensate area. (h) Comparison of average nucleosome crossing times across three groups: large condensates (N = 4 biological replicates), small condensates (N = 5 biological replicates), and molecules in which no condensates were detected (N = 4 biological replicates). Error bars represent standard deviation; p-values are from unpaired two-tailed t-tests. i, Nucleosome disassembly pathway obtained by performing successive pulling and relaxation cycles of a single nucleosome tethered between DNA handles attached to two optically trapped beads. The cycles were conducted in the absence (red), or presence of NDF (green), FACT (cyan), or both FACT and NDF proteins (yellow). The percentages of nucleosomes (N), hexasomes (H), tetrasomes (T), and bare DNA (D) observed after each cycle are presented. N = 50 biological replicates per condition.
