Extended Data Fig. 1: Additional DNA HiDRO spot efficiency and RNA HiDRO workflow, Related to Fig. 1.
From: High-throughput Oligopaint screen identifies druggable 3D genome regulators
a) Labelling efficiency for D1 locus as measured by percent of nuclei with at least one signal for different Oligopaint probe designs including dye-conjugated 80-mers (DC 80), secondary 80-mers (SL 80) and secondary labelled 42-mers (SL 42, conventional Oligopaints used in ref. 7). Each bar is mean +/− SD. [n = 12 biological replicate wells for all conditions except DC80 1pmol/well and 2pmol/well (n = 24) and UL80 25 pmol/well (n = 11)]. b) Labelling efficiency for D2 locus for different Oligopaint probe designs. Each bar is mean +/− SD. [n = 12 biological replicate wells for all conditions except DC80 1pmol/well and 2pmol/well (n = 24) and UL80 25 pmol/well (n = 11)]. c) Ideograms showing chromosomal locations of Oligopaint probes to 42 DNA regions tested by HiDRO. d) Labelling efficiency as measured by percent of nuclei in a well with one or more signals detected. Chr22 D1 (green) and D2 (magenta) highlighted. Each data point represents mean +/− SEM of six biological replicates. e) – (p) Hi-C contact matrices for boundaries tested by DNA HiDRO. Hi-C from ref. 4; tracks below are Oligopaint design, RAD21 ChIP-seq peaks (ENCODE ENCFF001UEG), CTCF ChIP-seq peaks with directionality (GEO GSM1022652), Genes, Compartment designation by eigenvector4, lamina associated domains (LADs) (4DN Data Portal: 4DNFI2BGIZ5F), superenhancers86, and insulation scores7. Percentages above each domain represent the probe efficiency for that domain as measured by the percentage of nuclei with at least one spot detected. q) Schematic for RNA HiDRO. Probes can be designed to introns and/or exons and RNA FISH is performed in 384-well plates. Wells are imaged on a high-content microscope, and nascent signals in the nucleus are segmented and measured computationally. Solid white line indicates nuclear edge. Scale bar field, 10 µm; Scale bar nucleus, 5 µm. r) Bursting frequency of three genes MCM5, LRCC20, and CHPF as measured by 3D RNA FISH on slides and RNA HiDRO shown. Each dot represents one biological replicate of bursting frequency calculated from greater than 100 nuclei. ns = P > 0.05, two-tailed t-test. [Biological replicate wells for MCM5 Slides and HiDRO: n = 4; LRCC20 Slides n = 6, HiDRO n = 4; CHPF Slides n = 6, HiDRO n = 10].
