Fig. 3

Description and validation of the RAT assay. a The flow diagram of the molecular biological part of the RAT assay. The light blue oval represents a region of the nucleus in the relative vicinity of a vlincRNA that would be co-purified with the vlincRNA by the RAT assay. The green and black lines represent DNA molecules that respectively are and are not located in the relative vicinity of vlincRNA. The red and purple lines represent specific oligonucleotides from the set 1 and 2 targeting each vlincRNA (short lines) and the cDNAs primed by these oligonucleotides (long lines). b An example of the DNA size distributions obtained after chromatin fragmentation in a typical RAT experiment for the DMSO- or drug-treated (etoposide or SN-38) samples. The assays performed with either the oligonucleotide set 1 (“P1”), 2 (“P2”), or the no-oligonucleotide control (“NP”) for the vlincRNA ID-1202. c Size distributions of particles obtained in a sorting experiment in the either the buffer (middle panel) and the buffer containing the chromatin fragmented using the conditions employed in a typical RAT experiment (bottom panel). The distribution of the particles with known sizes of 100, 200, and 300 nm is shown in the top panel. Note the increase in the fraction of the particles in the 300–500 nm range in the fragmented chromatin sample vs the sorting buffer (7.06% vs 2.85%). d The flow diagram of the analytical part of the RAT assay. e Top: definition of the odds ratio and the depiction of the hypothesis tested in the part below. Bottom: box plots of the odds ratios of the overlaps between the two biological replicas of the RAT assay at the gene (left) and region (right) levels at different RAT signal thresholds (X-axes)