title: GSM6640323: MP_product_repeat1; synthetic construct; OTHER
library_construction_protocol: Illumina adapter sequences were incorporated to the libraries using PCR. In high-throughput pri-miRNA cleavage assays, 3 pmol of each randomized pri-miRNA group were incubated with 5 pmol of the MP without or with 10 pmol of SRSF3 or SRSF7 at 37°C for 2 h, in 10 μL of the standard reaction buffer as mentioned above. After the reaction, the cleaved RNA products were purified using phenol extraction followed by isopropanol precipitation. The purified RNAs were ligated with the RA5-6N adapter (GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN NN) and then the resulting RA5-6N-ligated RNAs were reverse-transcribed using R-RA3 primer and SuperScript™ IV Reverse Transcriptase to generate cDNAs. The cDNAs were finally amplified using PCR with the sequencing primers into DNA libraries of the cleaved products. The pri-miRNA substrates were cloned using a circularization method. The pri-miRNAs were first reverse transcribed using SuperScript™ IV Reverse Transcriptase and cirRTP primer (5Phos/NNN NNN GAT CGT CGG ACT GTA GAA CTC TGA AC/iSp18/CCT TGG CAC CCG AGA ATT CCA). The resulting cDNAs were circularized by CircLigase™ ssDNA Ligase (Cat. No. CL4115K from Lucigen). The circularized cDNAs were eventually amplified by PCR using the sequencing primers into DNA libraries of the pri-miRNA substrates.