ATAC-seq on mouse embryonic stem cells for asynchronous cells, mitotic cells and purified mitotic chromosomes.

E14Tg2a mouse embryonic stem cells (ESCs) were cultured on gelatin coated plates in KO-DMEM medium supplemented with 15% FCS and 1000 U/ml leukaemia inhibitory factor. Rapidly dividing cultures of ESCs were arrested in metaphase using demecolcine (0.1 μg/ml for 6 h), achieving samples where most (85-90%) cells were in M-phase as judged by propidium iodide labelling. Condensed chromosomes were released using polyamine buffer, stained with Hoechst 33258 and Chromomycin A3 and purified by flow cytometry using a Becton Dickinson Influx equipped with specialised air-cooled lasers. ATAC-seq was performed in duplicate on nuclei from asynchronous cells (5x10^4) or directly on mitotic cells (5x10^4) or flow-sorted mitotic chromosomes (2x10^6). Libraries were sequenced on the Illumina NextSeq system (75 bp, paired end). ATAC-seq performed in duplicate in three conditions.