Fig. 4: Optimization and validation of ultrafast bisulfite conditions using human 28S rRNA and tRNA.
From: Ultrafast bisulfite sequencing detection of 5-methylcytosine in DNA and RNA
a, BS reaction of an RNA probe (5′-AGCGA) was monitored by MALDI-TOF MS. The peaks at 1,592, 1,675 and 1,593 were assigned to RNA oligo probes containing unconverted C, U-BS and final product U, respectively. As comparison, the RNA probe containing a m5C modification (5′-AGm5CGA) showed no visible reaction after 10 min of UBS-2 treatment. The peak at 1,606 represented unreacted m5C-containing oligo probe, and no peak representing the corresponding product was observed at 1689. b, Optimization of reaction time and temperature using 28S rRNA. The y axis is the average number of unconverted ratios for each condition based on two technical replicates (n = 2). The trade-off of the positive signal and the background noise indicated that 98 °C for 9 min is the best condition. c, The unconverted rate of all C and m5C sites along 28S rRNA. The detected m5C fractions at the two known m5C sites were over 95%, while no false positive (FP) was detected using the UBS-seq protocol when the detection threshold was set to 5% (FP% = 0). The original numbers for each site in two replicates (n = 2) are presented together. d, Comparison of the FP rate at non-m5C-modified C sites on ribosomal RNA (n = 1,690) among Zymo EZ RNA Methylation Kit, the three previously reported protocols8,9,35 and the UBS-seq condition (n = 2). e, Comparison of the detected fractions of the two m5C sites on ribosomal RNA using the same data in the panel (d). While the detected fractions at the m5C sites dropped dramatically in the previously reported protocols with prolonged treatment, UBS-seq detected high stoichiometry at these m5C sites. f, tRNA m5C sites detected in WT and NSUN2 KO cell lines (n = 2). The m5C sites deposited by DNMT2 were in orange and sites installed by NSUN2 were in red. The size of the dot represented the modification level in WT cells, and the y axis showed the change of m5C level upon NSUN2 KO. g, Converted ratio of C sites along tRNA AspGTC. The two replicates of the WT cells are on the left side, while the two replicates for NSUN2 KO cells are on the right side. C sites, DNMT2-targeted m5C sites and NSUN2-targeted m5C sites were colored in red, orange and green, respectively. FP, false positive.
