show Abstracthide AbstractWe apply ChIA-Drop, a single-molecule ligation-free mapping technique, to visualize loop formation over time. Our results demonstrated a cohesin-centric framework that coordinates with CTCF and RNAPII, respectively. At the loading (NIPBL binding) site, cohesin is highly correlated with transcriptional activities and translocates bi-directionally toward CTCF motif sites, where CTCF likely provides the anchoring point for cohesin to actively reel the chromatin template according to the motif forward orientation. Furthermore, cohesin specifically co-localizes with RNAPII and together mediates multiplex chromatin interactions that involve promoters, enhancers and super-enhancers. Intriguingly, single-molecule mapping data revealed that individual constituents of super-enhancers interact with target genes singularly and in cascade through intermediate regulatory elements, suggesting a probability-based mechanism for transcription regulation. Overall design: The microfluidics device (10X Genomics' Chromium instrument) produces Gel beads in Emulsion (GEM) with chromatin complexes for complex-specific DNA barcoding and amplification. Barcoded DNA amplicons from all GEMs (droplets) are pooled for DNA sequencing and mapping for identifying chromatin interactions. In the ChIA-Drop experiment, the microfluidics device (10X Genomics? Chromium instrument) produces Gel beads in Emulsion (GEM) with chromatin complexes for complex-specific DNA barcoding and amplification. Barcoded DNA amplicons from all GEMs (droplets) are pooled for DNA sequencing and mapping for identifying chromatin interactions. In the Hi-C experiment, the fragmented chromatin DNA was ligated after DNA ends was repaired with biotin labeled nucleotide. The ligated chromatin was sheared and then ligated with adaptors. The fragments were amplified after the ligated chromatin DNA was enriched by streptavidin beads. The chromatin interactions will be defined according to the mapping data of two ligated DNA fragments (tags). In the ChIA-PET experiment, the protocol is similar with Hi-C except two points: 1). The fragmented chromatin DNAs were ligated by biotin labeled 19bp linker rather than end repairment with biotin labeled nucleotide. 2). The ligated chromatin DNAs pulled down by antibody of interested protein through chromatin immunoprecipitation was tagmented by Tn5 transponase, which will be used for PET cluster amplification. In the ChIP-Seq experiment, the fixed chromatin was sheared by sonication and followed by chromatin immunoprecipitation with antibody of specific protein. The qualified ChIP-DNA will be used for library preparation to define the specific binding sites of target protein at the whole genome level. In the RNA-Seq experiment, total mRNA will be enriched for the library preparation. The whole transcriptome data will be used to reveal the presence and quantity of RNA expressed in the sample.