Extended Data Fig. 1: ChIA-Drop method optimization.

The efficiency of the microfluidics system for chromatin DNA barcoding and amplification was characterized by MiSeq sequencing data, in which each test generated 2–4 million sequencing reads. The numbers of captured GEM barcodes, the percentages of uniquely mapped reads, and the read-length distribution are presented for data quality assessment. a, Pure DNA versus chromatin DNA. Both pure DNA and chromatin DNA templates were prepared from the same chromatin sample. The chromatin sample was prepared by in situ HindIII digestion, followed by sonication for nuclear lysis. The chromatin DNA used for test was in a crosslinked state, and some DNA positions were bound by a protein component. The pure DNA was purified from the chromatin fragment after de-crosslinking. The length of the DNA templates was about 3,000 bp. Most of the pure DNA sequencing reads were of maximum length (130 bp); of these reads, 96% were mappable. The chromatin DNA yield 59% mappable reads. b, Distance density comparison of pure DNA and chromatin DNA. The relative probability densities of the log₁₀ of fragment-to-fragment distances in a GEM are plotted, categorized by the number of fragments per GEM (F#), colour-coded from blue to red for fragment numbers F = 2 to F = 11. Pure DNA (left) and chromatin DNA (right) data are both plotted on the same colour scale. The GEMs containing a low number of fragments showed distributions similar to those of the pure DNA, whereas the chromatin DNA with GEMs contain a high number of fragments displayed different patterns. c, Two-dimensional heat map comparison of pure DNA (HindIII, 6-bp cutter) and chromatin DNA (Mbol, 4-bp cutter or HindIII). The pure DNA data show random interactions and a lack of chromatin topological structures; the Mbol chromatin DNA data show some evidence for these structures, and HindIII chromatin DNA data show rich data for abundant chromatin contact structures. d, Chromatin fragment length by different fragmentation methods. Chromatin sample digested by a 4-bp cutter (MboI, ~ 300 bp) or a 6-bp cutter (HindIII, ~3,000 bp), or sheared by sonication (~ 6,000 bp) were prepared accordingly. The longer chromatin fragments (3,000–6,000 bp) generated a larger number of mappable DNA sequencing reads (≥50 bp) than did the shorter fragments. e, Summary statistics of GEMs from chromatin libraries prepared by Mbol and HindIII digestion. The read statistics between the two libraries are comparable under the same loading amount, but the fragment histograms of GEMs are different between the two libraries. The HindIII data generated a larger number of uniquely mappable reads, and of GEMs containing a high number of fragments than did the Mbol data, which contributes to differences seen between the chromatin structures shown in c. f, Chromatin sample loading by different input quantity. An input of 0.5 ng of chromatin DNA yields optimal results. When input was too low (0.5 pg), the majority of the sequencing reads were only 19–20 bp (barcode primer sequence) in length, which indicates that most droplets lack chromatin materials. g, Inter-species chromatin experiment. Chromatin samples of Drosophila S2 and human GM12878 cells were mixed in an equal number of cells or in an equal quantity of chromatin DNA. Barcoded sequencing reads were mapped to each reference genome. Reads with the same GEM barcode were grouped as a GEM. GEMs with fly-only, human-only or mixed reads were identified. The ratio of mixed GEMs to total GEMs provides an approximate likelihood of a mixed chromatin complex in a droplet. When tested with equally mixed numbers of cells, the number of GEMs with chromatin fragments of human origin is 20-fold more than the number of GEMs of Drosophila origin (181,956/9,149 = 19.89), which approximately reflects the ratio of the genome lengths for humans and Drosophila (3,000 Mb (human)/175 Mb (Drosophila) = 17.14). Notably, in the test with equal chromatin mass, the GEMs with mixed origins of fragments were only 5.1% of the total, which indicates that the proportion of droplets with mixed chromatin samples is small.