Extended Data Fig. 3: Analyses of MeRIP-seq/m6A-seq in HEK293T using Macs2.
From: Systematic calibration of epitranscriptomic maps using a synthetic modification-free RNA library
Three commercial antibodies (NEB[E1610S], SYSY [202003] and Abcam [ab190886]) are used for MeRIP-seq experiment. a) The metagene plot for transcriptome-wide distribution of each kind of peaks, including mRNA (peaks in cellular mRNA), calibrated (mRNA peaks excluding the false positive peaks), false positive (peaks in both cellular mRNA and IVT RNA), IVT (peaks in IVT RNA), and IVT unique (peaks explicitly present in IVT RNA). The location of peak summit is determined to represent the peak position. b) Cumulative frequency curves for the distance between each peak summit and nearest GGACH motif. c) The frequency of GGACH motif within the flanking 200 nt regions to the summit of each kind of peak. d) Nucleotide composition of immunoprecipitated oligonucleotides with randomized sequences (the central position is a determined A or m6A). e) The base ratio for the flanking 200 nt regions around the summit of detected peaks. Base ratio = the density of individual bases in peak region/that in transcript scope. Box boundaries represent 25th and 75th percentiles; the center line represents the median; whiskers indicate ±1.5× interquartile range (IQR). The same definition for box plot is also applied for panel (g). f) Two most significant motifs and the corresponding E-values for false positive peaks identified using three antibodies. g) The expression levels of genes with peaks are calculated separately from mRNA and IVT RNA. Two-sided t-test or paired t-test is applied to calculate significance. Correlations of expression levels are plotted for genes containing false positive peaks in mRNA and IVT RNA libraries.
