GSM9347623: EFO21 cells, DMSO, 24 h, rep2; Homo sapiens; OTHER
GLORI (glyoxal and nitrite-mediated deamination of unmethylated adenosines) sequencing was carried out as described in Liu et al., 2023. Total RNA was purified by adding Trizol directly to cultured cells and then extracting RNA using Qiagen miRNeasy columns according to the manufacturer's instructions including a DNase treatment step on column. Polyadenylated RNA was purified from 7.5 μg total RNA using the NEBNext PolyA mRNA magnetic isolation module (NEB E7490) according to the manufacturer's instructions but eluting in 18 μl nuclease-free water and fragmented by incubating (8 min, 94 °C) with 2 μl 10x NEBNext RNA fragmentation buffer. The fragmentation reaction was stopped by adding 2 μl 10x NEBNext RNA fragmentation stop solution and RNAs were purified using an RNA clean and concentrator column (Zymo, R1015) eluting in 14 μl. 1 ng of spike-in probe mix consisting of 5 different sequences each with an m6A modification site modified to 5, 20, 50, 80 or 100 % was added to each sample. To convert unmodified A positions to Gs, RNA was first incubated (30 min, 50 °C) with 6 μl glyoxal solution (8.8 M in H2O, Sigma, 50649-25 ml) and 20 μl DMSO and then incubated (30 min, 50 °C) with 10 μl saturated H3BO3 and finally incubated (8h, 16 °C) with 50 μl deamination buffer (750 mM NaNO2, 40 mM MES pH 6, 10 μl glyoxal solution [8.8 M in H20]). RNAs were precipitated by incubating (overnight, -80 °C) with 300 μl ethanol and 1.5 μl Glycoblue (ThermoFisher, AM9515) and centrifuging (20000g, 30 min, 4°C). RNA pellets were washed, dried, incubated (10 min, 95 °C) with 50 μl deprotection buffer (25 μl 1 M TEAA pH 8.6, 22.5 μl formamide) and purified using an RNA clean and concentrator column. RNA was incubated (30 min, 37 °C) with 5 μl T4 PNK buffer, 5 μl ATP, 1 μl Superase.In, 2 μl T4 PNK in a final 50 μl volume. RNAs were purified using an RNA clean and concentrator column, eluting in 6 μl, and generated into libraries for sequencing using the NEBNext multiplex small RNA library kit for Illumina according to the manufacturer's instructions. Final libraries were pooled and size-selected by gel electrophoresis selecting for bands from 160-250 bp. Libraries were sequenced to a depth of greater than 100 million PE150 reads on a NovaSeq X plus Illumina machine.