M 280 streptavidin magnetic bead

For the biotin-coupled probe pull-down assay, the biotinylated-circNHSL1 probe was specifically designed and synthesized through GenePharma (Shanghai, China), and oligo probe acted as a control. In brief, GC cells (1 × 10⁷) in lysis buffer were incubated with the designed probes for 2 h. Then, the biotin-coupled RNA complex was pull-downed by incubating the GC cell lysates with M-280 Streptavidin magnetic beads (Invitrogen). At 4 h post-incubation, the beads were washed with lysis buffer, and the RNA complexes bound to the beads were extracted with Trizol reagent (TaKaRa) and analyzed by RT-qPCR assay.
For biotin-coupled miRNA capture, GC cells (5 × 10⁶) were transfected with biotinylated miR-149-5p mimic (biotin-miR-149-5p, GenePharma) or nonsense control (biotin-NC, GenePharma) based on the user’s guidebook of Lipofectamine RNAiMax (Invitrogen). At 48 h after transfection, M-280 Streptavidin magnetic beads (Invitrogen) were washed with lysis buffer, followed by blockage with yeast tRNA on the rotator at a low speed (10r/min). After lysis and ultrasonic treatment, GC cells were incubated with the blocked beads at 4°C. The next day, Trizol reagent (TaKaRa) was used to purify bound RNAs, and RT-qPCR assay was conducted to assess the abundance of circNHSL1 in bound fractions.

Cells were transfected with biotinylated circ_0038138 (WT), circ_0038138 (MUT) and NC probes synthesized by Guangzhou RiboBio Co., Ltd. (Guangdong, China). The cell lysates were then incubated with M-280 streptavidin magnetic beads (Invitrogen Inc., Carlsbad, CA). Magnetic RNA extraction kit (A27828, Thermo Fisher Scientific) was applied to perform RNA extraction and analysis according to the supplier's instructions. Cells were transfected with a biotinylated probe, and the cell lysates were incubated with the M-280 streptavidin magnetic beads (Invitrogen) [26] (link). The expression of miR-198 was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) (Supplementary Table 1).