Extended Data Fig. 2: Detecting immunogenic RNAs that are induced after treatment with 5-AZA-CdR using MDA5/RNase-protection assay.

a, MDA5/RNase-protection assay experimental scheme created with BioRender.com. To identify the primary ligand for MDA5, total cytosolic RNA (5 ng μl⁻¹) was purified from mock or 5-AZA-CdR treated (300 nM for 5 days) patient-derived CRC cells and pre-incubated with MDA5-∆2CARD protein, followed by either RNase A (+RNase A) digestion or without RNase A digestion (−RNase A). The remaining RNA was then purified and sequenced. b, Venn diagram showing the number of IR-Alu pairs at the baseline (n = 1,040 IR-Alu pairs), immunogenic (5-AZA-CdR) (n = 4,482 IR-Alu pairs) and treatment-induced (n = 3,687 IR-Alu pairs) conditions. c, Bar plot showing the number of baseline immunogenic RNA (IR-Alu pairs = 1,040, non-IR Alu pairs = 97). d, Bar plot showing the number of treatment-induced immunogenic RNA (IR-Alu pairs = 3,687, non-IR Alu pairs = 547). e, f, Average reads per million (RPM) profile of baseline MDA5-protected inverted-repeat transcripts (n = 1,176 transcript regions) (e) and treatment-induced MDA5-protected inverted-repeat transcripts (n = 3,895 transcript regions) (f). The box plots include the RPM scores of these transcripts. P value was calculated using a two-sided Wilcoxon signed-rank test. Box plot statistics containing the minimum, first quantile, median, third quantile, and maximum are 4.76, 10.21, 15.15, 27.44, and 52.53, respectively, for the mock-treated sample, and 0.0, 7.69, 15.24, 29.96, and 63.35 for the 5-AZA-CdR-treated sample in e, and 0.00, 0.79, 2.976, 6.185, and 14.26 for the mock-treated sample, and 2.41, 5.47, 7.47, 10.98, and 19.22 for the 5-AZA-CdR-treated sample in f. g, Schematic representation of inverted-repeats intramolecular pairing versus sense and antisense transcription created with BioRender.com. Inverted repeats can form intramolecular stem–loops when one strand is transcribed, as well as intermolecular duplexes when both strands are transcribed.