Phase-Separated NDF-FACT Condensates Facilitate Transcription Elongation on Chromatin

Description

This dataset contains single-molecule nucleosome transcription and nucleosome unwrapping data used in Li & Burgos-Bravo et al., 2025 (Nature Cell Biology). The study investigates how the transcription elongation factors NDF and FACT cooperate to facilitate RNA Polymerase II (Pol II) progression through nucleosomal barriers via formation of phase-separated condensates. Using high-resolution dual-trap optical tweezers and the Lumicks C-Trap instrument (integrating optical tweezers with confocal fluorescence detection), we collected real-time transcription trajectories and nucleosome unwrapping/rewrapping traces in the presence or absence of NDF and/or FACT. The source data includes: - Raw transcription traces of Pol II transcribing through a nucleosome, used to quantify Pol II residence time (Fig. 3B, 3C, Extended Data Fig. 3A). - Raw transcription traces and confocal fluorescence scans obtained from the C-Trap instrument and used to visualize condensate formation during transcription at the single-molecule level (Fig. 3E, 3F, and Extended Data Fig. 3C, 3E, 3F). This includes both the dataset shown in the manuscript and additional representative traces not included in the figures. - Raw force-extension curves showing nucleosome disassembly products across successive pulling cycles (Extended Data Fig. 3I).

Steps to reproduce

Folder Raw Transcription Traces_Figure 3B Each folder contains raw transcription traces (.mat files) of individual Pol II molecules transcribing through a single nucleosome, either alone or in the presence of transcription factors (FACT and/or NDF). These traces were analyzed using custom-written MATLAB code, as described in Burgos-Bravo et al., 2025 Molecular Cell (10.1016/j.molcel.2025.05.002). The analysis code is publicly available at Zenodo: https://doi.org/10.5281/zenodo.3718873 and is used for: I. Converting QPD voltage signals into base pair (bp) distances II. Aligning traces to improve precision in determining Pol II position along the DNA template III. Generating residence time histograms   Folder Raw Transcription Traces + Fluorescence_Figure 3E, 3F and Extended Data Fig. 3C, 3E, 3F Each folder contains the raw data (.mat and .tiff files) used to generate the corresponding figure panel; Figure 3E, 3F, and Extended Data Fig. 3C, 3E, 3F. Specifically, each folder includes two files: I. Raw transcription trace (.mat): Contains force, distance, time, and photon count data acquired using the Lumicks C-Trap instrument, which integrates dual-trap optical tweezers with confocal fluorescence microscopy. Raw transcription traces were analyzed using the same custom-written MATLAB code used for Figure 3B II. Scan file (.tiff): Confocal scan image used to generate the kymograph, processed using Fiji. Folder Raw Transcription Traces + Fluorescence_Figure 3E, 3F and Extended Data Fig. 3C, 3E, 3F_OtherExamples_NotInPaper This folder contains additional representative transcription and fluorescence traces corresponding to the experimental conditions shown in Figures 3E, 3F, and Extended Data Fig. 3C, 3E, 3F of the manuscript. These example traces were not included in the final figures but illustrate similar behaviors and support the conclusions presented. However, they were used to prepare Extended Data Fig. 3G, 3H. Folder Raw Nucleosome unwrapping-rewrapping traces_Extended Data Fig. 3I Each folder contains raw force-extension traces (.mat files) of nucleosome unwrapping/rewrapping events in the absence or presence of transcription factors (FACT and/or NDF). Each trace includes the 1st, 2nd, 3rd, and 4th pulling cycle events. The same custom-written MATLAB code used for the analysis in Figure 3B was used to analyze these pulling traces.

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