GSM8149348: SNU719, rep2; Homo sapiens; OTHER
SNU719 and AGS cells were seeded in 15 cm plates to achieve 80% confluence the next day. Cells were transfected with 10 μg of the MPRA plasmid library using TransIT-X2 reagent (Mirus Bio, MIR 6004) as per the manufacturer's protocol, aiming for a transfection efficiency of 50-80%. Total RNA was collected 48 hr post-transfection using the miRNeasy mini kit (Qiagen, #217004). Genomic DNA was removed using the Turbo DNA-free kit (ThermoFisher Scientific, AM1907) following the manufacturer's “Rigorous DNase treatment” protocol. Per replicate, 15 μg total RNA was reverse transcribed with SuperScript IV Reverse Transcriptase (ThermoFisher Scientific, #18090010) using oligo-dT primers. Amplicon sequencing libraries from cDNA or plasmid pool DNA were constructed through two PCR reactions. In the first PCR round, 1:10 diluted cDNA was amplified using 0.2 μM of the DT_barcodePE_Fv2 and 0.2 μM of an isomolar mix of the DT_barcodePE_Rv2 primers (0 to 6 random Ns, Table S2). The PCR reaction was performed with the NEB Next High-Fidelity 2x PCR Master Mix (NEB, M0541L) and the following conditions: 98C for 30 sec, 10 cycles (98C for 10 sec, 63C for 10 sec, 72C for 15 sec), 72C for 2 min. Enough PCR reactions were run to ensure that all the cDNA from each replicate was amplified. In the second PCR reaction, 1:10 diluted PCR round 1 product was amplified using 0.5 μM of a unique pair of multiplexing Illumina primers (PE_i5 and PE_index in Table S2). The following PCR conditions were used: 98C for 30 sec, 10 cycles (98C for 10 sec, 62C for 10 sec, 72C for 15 sec), 72C for 2 min. For each replicate, the second round PCR product was purified through gel extraction using the Monarch Gel Extraction kit (NEB, T1020S). The quality of each library was assessed by an Agilent Tapestation D1000 assay (Agilent). An equimolar mix of all libraries was sent for single-end 150 bp sequencing on an Illumina sequencer, with 20% PhiX spike-in to increase library complexity.