Extended Data Fig. 6: Systematic optimization and benchmarking of A3A deamination reactions in snhmC-seq2.
From: Joint single-cell profiling resolves 5mC and 5hmC and reveals their distinct gene regulatory effects
(a) Boxplot illustrating a comparison of lambda spike-in 5mCG (left) or CH (right) non-conversion rate between snhmC-seq and snhmC-seq2, with mean values below each plot (in red). The boxes display the median (center line) and interquartile range (from the 25th to 75th percentile), the whiskers represent 1.5 times the interquartile range, and open white diamonds indicate the mean value. (b) SDS-PAGE analysis of the concentration and purity of both in-house wild-type A3A and New England Biolabs (NEB) A3A* enzymes. Shown is a representative result from two independent batches of in-house A3A purification using the improved pET-His6-MBP-A3A-Gly6His6 vector (Addgene, catalog no. 187822) and PreScission protease cleavage strategy. Both bathes show similar purity and total yield. The expected molecular weight of MBP-A3A-His (before cleavage) is ~70 kD, while that of untagged A3A is ~24 kD. Bovine serum albumin (BSA, ~66kD) serves as control to quantify the concentration of proteins. (c) Box plots illustrating deamination failure rate (top panel, 5mCG context; bottom panel, CH context) on in vitro CpG-methylated lambda phage gDNA across different concentrations of both NEB A3A* (orange) and in-house A3A (blue), with mean values below each plot. Open white circles indicate mean failure rates (indicated in red). The box plot elements are defined as in a. The number of nuclei (passed sequencing quality control filter) tested in each condition is shown on top.