Dissecting gene activation and chromatin remodeling dynamics in single human cells undergoing reprogramming

During cell fate transitions, cells remodel their transcriptome, chromatin, and epigenome; however, it has been difficult to determine the temporal dynamics and cause-effect relationship between these changes at the single-cell level. Here, we employ the heterokaryon-mediated reprogramming system as a single-cell model to dissect key temporal events during early stages of pluripotency conversion using super-resolution imaging. We reveal that, following heterokaryon formation, the somatic nucleus undergoes global chromatin decompaction and removal of repressive histone modifications H3K9me3 and H3K27me3 without acquisition of active modifications H3K4me3 and H3K9ac. The pluripotency gene OCT4 (POU5F1) shows nascent and mature RNA transcription within the first 24 h after cell fusion without requiring an initial open chromatin configuration at its locus. NANOG, conversely, has significant nascent RNA transcription only at 48 h after cell fusion but, strikingly, exhibits genomic reopening ..., The dataset contains quantifications related to STORM, OligoSTORM, and smRNA-FISH data.
Cell lines and culture conditions
Primary human BJ fibroblasts (ATCC) were routinely plated at a density of 5x103 cells/cm2 and maintained in DMEM 1X (Gibco) containing 10% of Fetal Bovine Serum (Cytiva), Penicillin/Streptomycin 1X (Gibco) and Sodium Pyruvate 1X (Gibco). Culture media was changed every other day. Cells were passaged every three to four days (until reaching 80-90% of confluency) using 0.05% Trypsin-EDTA (Gibco). Tcf3-/- mouse embryonic stem cells (mESCs)1 at passages 25-28 were plated at a seeding density of 15-20x104 cells/cm2 and cultured in KnockOutTM DMEM, 1X (Gibco), containing 15% of Fetal Bovine Serum (Cytiva), Penicillin/Streptomycin 1X (Gibco), Sodium Pyruvate 1X (Gibco), GlutaMAX 1X (Gibco), MEM aminoacids 1X (Gibco), 2-Mercaptoethanol 50 µM (Gibco) and 106 units/ml of LIF (Leukemia Inhibitory Factor, EMD Millipore). Media was changed daily and cells were passaged every thre..., , # Data from: Dissecting gene activation and chromatin remodeling dynamics in single human cells undergoing reprogramming

.txt files containing quantifications related to the research manuscript \"Dissecting gene activation and chromatin remodeling dynamics in single human cells undergoing reprogramming\"

The dataset is divided into different text files, each one corresponding to the quantifications shown in the plots inside the panels of the Figures or Supplementary Figures of the manuscript.

Software used for super-resolution data analysis is found in:

[https://github.com/melikelakadamyali/Martinez-Sarmiento_et_al_2023](https://github.com/melikelakadamyali/Martinez-Sarmiento_et_al_2023)

[https://github.com/melikelakadamyali/StormAnalysisSoftware](https://github.com/melikelakadamyali/StormAnalysisSoftware)

Additional software used to process images

FISH-QUANT
[https://doi.org/10.1038/nmeth.2406](https://doi.org/10.1038/nmeth.2406)

ImageJ
[https://imagej.net/ij/index.html](https://...