Mouse embryos were dissected at embryonic day (E) 8.25 in 1X PBS + 2% FCS and placed individually onto a parafilm strip. Overflowing liquid was removed and each parafilm strip was inserted into a cryovial, which was subsequently snap-frozen in liquid nitrogen. Frozen cryovials were stored at -80C. For single-nucleus ATAC-seq, 10 mouse embryos were thawed, pooled and processed using the snATAC-seq protocol with slight modifications. Briefly, after permeabilization with OMNI buffer, 1,500 nuclei were placed per well in two 96-well plates. Nuclei were then tagmented with Tn5 to introduce a first barcode (T7), as in (Preissl et al., 2018), and pooled into one tube. Nuclei were then stained with DRAQ7 (1:200; Cell Signaling Technology #7406; LOT# 31DR71000) for 5 min, and 40 nuclei per well were subsequently sorted into six 96-well plates using SH800 sorter, and a second barcode (i5) was introduced by PCR. During the sort, two populations corresponding to small and large size nuclei were detected. To make sure nuclear size did not affect subsequent bioinformatics analysis, we sorted two extra plates, where small and large nuclei were specifically sorted. You will see that sample titles contain "all", "large" and "small" in their names. "all" refers to the FACS gating where nuclei were sorted indiscriminately; "large" refers to the FACS gating strategy to sort for large size nuclei; "small" refers to the FACS gating strategy to sort for small size nuclei. Libraries were sequenced using three NextSeq500, 50 paired-end sequencing runs, and one HiSeq2500 100 paired-end run. Nuclei were tagmented with Tn5 in two 96 well-plates to introduce a first barcode (T7). After pooling, 40 nuclei were sorted into each well of six 96-well plates and a second barcode (i5) was introduced by PCR. Libraries were sequenced on a HiSeq2500 or NextSeq500 (Illumina) with following read lengths: 50 + 10 + 12 + 50 (Read1 + Index1 + Index2 + Read2). Index1 corresponded to the T7 barcode and Index2 corresponded to the i5 barcode.