We quantified the expression of bidirectional loci for each strand and 200 bp flanking window in each of the 432 primary cell, 135 tissue and 241 cell line samples separately by counting the CAGE tags whose 5' ends were located within these windows. The expression values of both flanking windows were normalized by converting tag counts to tags per million mapped reads (TPM) and further normalization between samples was done using the RLE normalization procedure in edgeR41. The number of CAGE tags aligned on ChrM was subtracted from the total number of aligned CAGE tags in each library before normalization. The normalized expression values from both windows were used to calculate a sample-set wide directionality score, D, for each enhancer over aggregated normalized reverse, R, and forward, F, strand expression values across all samples (Supplementary Fig. 6a); D = (F−R) / (F+R). D ranges between −1 and 1 and specifies the bias in expression to reverse and forward strand, respectively (D=0 means 50% reverse and 50% forward strand expression, while abs(D) close to 1 indicates unidirectional transcription). A directionality score
