Assessing cell to cell, and importantly, chromosome to chromosome, heterogeneity in cellular phenotypes is a central goal of modern cell biology. However, chromatin profiling in single cells has been extremely challenging, and single chromosome profiling has not been achieved. In cases where single cell methods have shown promise, success has been mainly limited to histone proteins and/or require highly specialized equipment or cell type specific protocols and are relatively low throughput. Here, we have combined the advantages of tagmentation, linear amplification and combinatorial indexing to produce a high throughput single cell DNA binding site mapping method that does not require specialized equipment and is capable of multiplexing several samples/ target proteins in one experiment. Targeted Insertion of Promoters (TIP-seq) uses Tn5 fused to protein A (as with CUT&Tag) to insert a T7 RNA polymerase promoter into sites adjacent to an antibody bound to a chromatin protein of interest, followed by linear amplification of flanking DNA with T7 polymerase, cDNA preparation and PCR indexing. Tip-seq provides ∼10-fold higher unique reads and thus higher coverage per single cell compared to state-of-the-art methods. We apply TIP-seq to map histone modifications, RNA PolII and CTCF binding sites in single human and mouse cells. TIP-seq will also be adaptable for other platforms, such as 10X genomics and ICELL8. In summary, TIP-seq provides a high-throughput, low-cost method for single cell protein mapping, that yields substantially higher coverage per cell and signal to noise than existing methods.

SUMMARYThe temporal order of DNA replication (replication timing, RT) is highly coupled with genome architecture, but cis-elements regulating spatio-temporal control of replication have remained elusive. We performed an extensive series of CRISPR mediated deletions and inversions and high-resolution capture Hi-C of a pluripotency associated domain (DppA2/4) in mouse embryonic stem cells. Whereas CTCF mediated loops and chromatin domain boundaries were dispensable, deletion of three intra-domain prominent CTCF-independent 3D contact sites caused a domain-wide delay in RT, shift in sub-nuclear chromatin compartment and loss of transcriptional activity, These "early replication control elements" (ERCEs) display prominent chromatin features resembling enhancers/promoters and individual and pair-wise deletions of the ERCEs confirmed their partial redundancy and interdependency in controlling domain-wide RT and transcription. Our results demonstrate that discrete cis-regulatory elements mediate domain-wide RT, chromatin compartmentalization, and transcription, representing a major advance in dissecting the relationship between genome structure and function.

In mammalian cells, distinct replication domains (RDs), corresponding to structural units of chromosomes called topologically-associating domains (TADs), replicate at different times during S-phase [1][2][3][4] . Further, early/late replication of RDs corresponds to active/inactive chromatin interaction compartments 5,6 . Although replication origins are selected stochastically, such that each cell is using a different cohort of origins to replicate their genomes [7][8][9][10][11][12] , replication-timing is regulated independently and upstream of origin selection 13 and evidence suggests that replication timing is conserved in consecutive cell cycles 14 . Hence, quantifying the extent of cell-to-cell variation in replication timing is central to studies of chromosome structure and function. Here we devise a strategy to measure variation in single-cell replication timing using DNA copy number. We find that borders between replicated and un-replicated DNA are highly conserved between cells, demarcating active and inactive compartments of the nucleus.Nonetheless, measurable variation was evident. Surprisingly, we detected a similar degree of variation in replication timing from cell-to-cell, between homologues within cells, and between all domains genome-wide regardless of their replication timing.These results demonstrate that stochastic variation in replication timing is independent of elements that dictate timing or extrinsic environmental variation.peer-reviewed) is the author/funder. All rights reserved. No reuse allowed without permission.The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/158352 doi: bioRxiv preprint first posted online Jun. 30, 2017; Dileep and Gilbert, page 3Single-cell DNA copy number can distinguish replicated DNA from un-replicated DNA 15,16 . Specifically, regions that have completed replication will have twice the copy number compared to regions that have not replicated. Hence, we reasoned that measurements of DNA copy number in cells isolated at different times during S-phase could reveal replication-timing programs in single-cells. Moreover, to separately evaluate the extent of extrinsic (cell-to-cell) vs. intrinsic (homologue-to-homologue) variability in replication timing, we examined both haploid H129-2 mouse embryonic stem cells (mESCs) and diploid hybrid musculus 129 × Castaneus mESCs that harbor a high single nucleotide polymorphism (SNP) density between homologues, permitting allele specific analysis. To generate single-cell copy number variation (CNV) profiles, we used flow cytometry of DNA-stained cells to sort single S-phase cells into 96 well plates followed by whole genome amplification (WGA). Amplified DNA from each cell was uniquely barcoded and sequenced (Fig. 1a) 17,18 . To control for amplification and mappability biases, we also sorted G1 and G2 cells, which contain a uniform DNA content. Regions of low mappability and over amplification were removed based on the G1 and G2 controls. Read counts were normalized by dividing the co...

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