The section provides detailed information on the modifications to the CLASH (Cross-Linking and Annealing for Sequencing of Transcripts) protocol and read statistics, as well as experimental validation of miRNA targets. Table S1 lists the internal identifiers and key differences in the experimental protocols for six CLASH experiments (E1-E6) compared to experiment E4. Experiments E7-E10 were conducted to assess background noise by adding yeast lysates to crosslinked HEK cell lysates. Table S2A and S2B provide read statistics, including the number of non-chimeric and chimeric reads, respectively. Non-chimeric reads (single reads) mapping to various RNA species account for about 70% of all reads, while chimeric reads, which represent about 2% of all cDNAs, are primarily associated with interactions between miRNAs and other RNA species. The mixed human-yeast lysate experiment was used to estimate background noise from RNA-RNA interactions after cell lysis. Table S3 lists experimentally validated miRNA targets found in the CLASH dataset, with about 40% of validated interactions supported by a single chimeric read. Table S4A and S4B show the overlap between targets identified in CLASH, CLASH single read clusters, PAR-CLIP clusters, and predictions from common algorithms. The enrichment of CLASH target predictions ranged from 12 to 97-fold, with a combined enrichment of 13.6-fold. Table S5 identifies motifs enriched in CLASH targets of individual miRNAs, and Table S6 provides a list of oligonucleotides used in the experiments.The section provides detailed information on the modifications to the CLASH (Cross-Linking and Annealing for Sequencing of Transcripts) protocol and read statistics, as well as experimental validation of miRNA targets. Table S1 lists the internal identifiers and key differences in the experimental protocols for six CLASH experiments (E1-E6) compared to experiment E4. Experiments E7-E10 were conducted to assess background noise by adding yeast lysates to crosslinked HEK cell lysates. Table S2A and S2B provide read statistics, including the number of non-chimeric and chimeric reads, respectively. Non-chimeric reads (single reads) mapping to various RNA species account for about 70% of all reads, while chimeric reads, which represent about 2% of all cDNAs, are primarily associated with interactions between miRNAs and other RNA species. The mixed human-yeast lysate experiment was used to estimate background noise from RNA-RNA interactions after cell lysis. Table S3 lists experimentally validated miRNA targets found in the CLASH dataset, with about 40% of validated interactions supported by a single chimeric read. Table S4A and S4B show the overlap between targets identified in CLASH, CLASH single read clusters, PAR-CLIP clusters, and predictions from common algorithms. The enrichment of CLASH target predictions ranged from 12 to 97-fold, with a combined enrichment of 13.6-fold. Table S5 identifies motifs enriched in CLASH targets of individual miRNAs, and Table S6 provides a list of oligonucleotides used in the experiments.