Dynabeads myone streptavidin c1 kit

RIP experiments were performed by using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA) following the manufacturer’s instructions. Immunoprecipitation of AGO2 was performed in QM7 cells co-transfected with circRBFOX2s expression vector and miR-1a-3p°, miR-206 or miR-203 control. The mRNA levels of circRBFOX2s were quantified by qRT–PCR and were normalized to GAPDH gene. The relative immunoprecipitate/input ratios are plotted.
The 3’ end biotinylated miR-1a-3p°, miR-206 or miR-203 mimic (RiboBio) were transfected into QM7 cells along with circRBFOX2s expression vector. The biotin-coupled RNA complex was pull-downed by Dynabeads MyOne Streptavidin C1 kit (Invitrogen) following the manufacturer’s instructions. RNA bound to the beads (pull-down RNA) was isolated using Trizol LS reagent (Invitrogen). The mRNA levels of circRBFOX2s in the streptavidin captured fractions were quantified by qRT–PCR and the enrichment ratios of the miR-1a-3p° or miR-206 to the miR-203 control were plotted.

The 3′-end biotinylated miR-146a mimics or the miRNA control (50 nm, General Biosystems, Anhui, China) was transfected into human primary keratinocytes for 48 h. Next, the cell lysates were harvested. Then Dynabeads MyOne Streptavidin C1 kit (Invitrogen, USA) was applied to enrich the biotin-coupled RNA combination. To remove unbound materials, the beads were pelleted and washed. The TRIzol reagent was used to extract RNA that was bound to beads. qRT-PCR was used to test the abundance of hsa_circ_0102678 in the isolated fractions. All sequences were listed in online supplementary Table 5.