Extended Data Fig. 7: Micro-C reveals distinct spatial organization of nucleosome arrays targeted by OSK.

a, Agarose gel electrophoresis showing gradual chromatin digestion with increasing amounts of MNase concentrations. 15 and 20 U of MNase (dotted red box) were used for Micro-C experiments. Representative image from n = 4 biological replicates, which were pooled together for sequencing. b, Profile plot showing mono- and di-nucleosome DNA fragment sizes before (top) and after (bottom) proximity ligation. c, Decaying curves of inter-nucleosomal Micro-C contacts zoomed within 200 bp and 2 kb distance. Micro-C contact density normalized by sequencing depth. Three curves showing distinct read pair orientations relative to one another are colour coded as shown in the schematics above. Contacts of up to six nucleosomes can be resolved (dashed line). Schematics illustrating the inter-nucleosomal contacts between n/n + x in different orientations are indicated on top. Insets show an example of n/n + 1 and n/n + 5 (painted blue) in 3’-to-5’ orientation. d, Micro-C pileup heatmaps of OSK nucleosome arrays in early reprogramming (top) and fully reprogrammed cells (bottom). Maps are plotted at 100 bp resolution for fine-scale inter-nucleosome contacts and centred around the upstream near-border. Yellow arrowheads indicate strong interactions between the near and far-border, which disintegrate in final reprogramming. e, OSK bind to more interactive enhancers after reprogramming. Pileup Micro-C analysis showing long-range interactions at 2 kb resolution around nucleosome arrays midpoints targeted by OSK in early (left) and final reprogramming (right). f, Circos plots showing long range interactions linking OSK binding (ChIP-seq) along with chromatin accessibility (ATAC-seq) and nucleosome positions (MNase-seq). More long-range interactions are observed after reprogramming. g, Reverse-phase HPLC analysis of chromatin histone extracts purified from MEFs (black), MEF-empty (grey), MEF-H1.4KD (red), and MEF-H1.4OE (green), showing the abundance of H1 variants. Absorbance at 214 nm in milli-absorbance-units (mAU) was plotted as function of elution time (min). h, Bar plots of relative H1 amounts quantified by HPLC in (g) showing the compensation effects of H1 variant expression after H1.4KD and H1,4OE in MEFs. i, LC-MS successfully deconvoluted the H1.3 (black) and H1.4 (grey) amounts in MEFs, MEF-empty, MEF-H1.4KD, and MEF-H1.4OE, which were not resolved by HPLC in (g). j, Western blot analysis showing the amounts of total H1 (using pan-H1 antibody) in MEF-H1.4KD and MEF-H1.4OE compared to MEF-empty. Protein ladder sizes in KDa are indicated. representative image from n = 5 biological replicates. Raw blots are shown in Supplementary Fig. 1. k, Average profile plots (top) and read density heatmaps (bottom) of ATAC-seq signal around nucleosome arrays bound by OSK in MEFs, MEF-empty, MEF-H1.4KD and MEF-H1.4OE. The nucleosome arrays were ranked ordered based on size and grouped into open and closed according to ATAC-seq in MEFs. The number of nucleosome arrays (n) are indicated. Four biological replicates (n = 4) were sequenced and merged for analysis. l, Experimental flow chart of ATAC-seq to measure the effects of H1.4KD (top) and H1.4OE (bottom) on chromatin accessibility as a proxy for OSKM binding during reprogramming early of TNG-KOSM-MEFs. Fully reprogrammed KOSM-MEFS were assessed by the expression of GFP, which has been knocked-in to one of the Nanog alleles (TNG).