Using the cDNAs from the transfected cells, the miRNA binding sites within the 3′ UTR of the luciferase genes were amplified in 50 μL PCR reactions using 0.3 μM flanking universal primer sand 25 μL 2X CloneAmpTM HiFi PCR premix (Takara, Mountain View, CA, United States). In a separate reaction for each sample, 2 μL of cDNA and 1 pg of the input plasmid pool was used as PCR template. PCR conditions used were: 98°C (10 s), 54°C (5 s), and 72°C (5 s) for 25 cycles. A 6-nucleotide unique molecular barcode was added to the 5′-end of both the forward and reverse primer (see Supplementary Table 2). The input pools (n = 4 replicates) and the five biological replicates in the three different cell lines were each ‘barcoded’ by a unique pair of sequences. The barcoded PCR products were purified using a MinElute® PCR Purification kit (Qiagen, Germantown, MD, United States). The barcoded libraries were combined in equimolar concentrations to create a sequencing pool with 19 different molecular barcodes. A schematic representation of the steps involved in creating this sequencing pool is shown in Figure 1.
