Extended Data Fig. 1: RCMC efficiently and reproducibly captures ligated dinucleosomal fragments, giving rise to deep contact maps.
From: Region Capture Micro-C reveals coalescence of enhancers and promoters into nested microcompartments
(a) Representative MNase titration DNA gel indicating the ideal level of digestion by MNase, based on the ratio of fragment sizes, for the RCMC protocol. (b) Representative size-selection gel for the RCMC protocol showing the dinucleosomal band that is extracted to obtain ligated fragments. (c) Overview of the capture probe design workflow for RCMC. 80-mer probes tiling the region of interest are designed, removing those which overlap highly repetitive regions. (d) Summary of the capture efficiency for each of the five regions for which probes were designed. The locations and sizes of the regions, the number of ligated fragments which mapped at single loci at both ends in total and in the region, and the capture efficiencies are given. Because different capture probe sets were used for Biological Replicates 1 (two separate sets of capture probes) and 2 (simultaneous capture for all five loci), numbers are separately provided for each Biological Replicate. (e) Contact maps comparing raw, unbalanced data (upper panel, lower triangle), ICE-balanced30 to all aligned reads (lower panel, lower triangle) and ICE-balanced to reads in captured loci only (both panels, upper triangle). Balancing only to data entirely within captured loci was necessary to remove artifacts due to capture bias. (f) Contact maps comparing the entire Fbn2 TAD in RCMC and in Hi-C31 and Micro-C12. Gene annotations and ChIP-seq signal tracks are shown below the contact maps. (g) Measurement of reproducibility between WT replicates across all five capture loci, with reproducibility scores determined using HiCRep58 at 10 kb resolution, clustered according to similarity. Three technical RCMC replicates (denoted by ‘TR#’) comprise Biological Replicate 1, while ‘BR2’ denotes Biological Replicate 2. TR3_WT is noted in blue text at the Sox2 and Nanog loci because very little TR3_WT pre-Capture library remained for input to Sox2 & Nanog capture after the initial Ppm1g, Klf1 and Fbn2 capture experiment; accordingly, relative to all other replicates, TR3_WT has much lower sequencing depth (0.5–2.4% the number of unique contacts) at the Sox2 & Nanog loci.
