Magnetic bead

Cells were washed thrice with PBS and then lysed on ice. The lysate was centrifuged at 1200 rpm for 15 min to remove the precipitate. The supernatant was divided into three groups (positive control, negative control, and experimental group): the positive control was directly frozen for use; IgG was added to the negative control, and IP antibody was added to the experimental group (TRIP12, ab86220; HACE1, #730103; Thermo Fisher Scientific, USA), and then incubated overnight at 4°C. Then, 40-μL magnetic beads (Thermo Fisher Scientific) were added to each group, and after shaking at 4 °C for 1 h, the magnetic beads were washed five times with precooled PBS, after which protein loading buffer was added, and the target protein was detected using western blotting.

An RNA Pulldown Kit (Geneseed, Guangzhou, China) was used to determine the binding efficiencies between circDNMT1 and miRNAs. The biotin-labeled circDNMT1 probe was mixed with magnetic beads (Thermo Fisher, USA) at room temperature for 2 h. The lysates of HGC-27 and AGS cells were prepared using lysis buffer and sonication, followed by incubation with probes at 4°C for 8 h. The magnetic beads were washed by washing buffer three times. The miRNAs were extracted by Trizol and were analyzed by qRT-PCR. The probe sequences can be viewed in Table S2.

The MEGAscript™ T7 High Yield Transcription kit (Invitrogen, USA) was used to transcribe biotin-labelled RNAs in vitro. Bio-16-UTP (10 mM, Ambion) was used for transcription. After adding 2 µL of Dnase I, the Eppendorf tube was incubated at 37 °C for 15 min to remove the DNA, then 2 µL of 0.2 M EDTA (pH 8.0) was added. In order to allow the RNA to form secondary structure, 1 µg of biotinylated RNA in RNA structure buffer was heated at 95 °C for 2 min, put on ice for 3 min, then left at room temperature for 30 min. Magnetic beads (Invitrogen, USA) were used to bind and enrich the RNAs. Folded RNA was then mixed with cytoplasmic extract from liver cancer cells in 500 µL RIP wash buffer. The Magnetic beads were re-suspended in 50 µL RIP wash buffer, then the suspension was added to Dynabeads M-280 Streptavidin (60210, Invitrogen) and incubated at 4 °C. The suspension was centrifuged for 1 min, and the supernatant was discarded. Magnetic beads were washed briefly with RIP wash buffer for six times and boiled in SDS buffer. The retrieved proteins were detected by western blot and mass spectrometry. RNA probes were as follows: lnc-Ma301 sense: taatacgactcactatagggGGAGAGTTTGGGTCACAGGAGC, lnc-Ma301 antisense: CCTACTTGTTTTTTTTATTTTGG.

The circNFIX probe was designed by SCbio (Guangzhou, China). SW1353 cells were transfected with circNFIX plasmids. After 24 h, cells were collected and lysed with 1 ml of lysis buffer and irradiated with 254 nm ultraviolet light. The circNFIX probe and Lac Z probes (control probes) mixed with magnetic beads (Life Technologies) were incubated at 25°C for 2 h. Then, the probe‐coated beads were incubated with the cell lysates at 4°C overnight. Non‐specifically bound RNAs were removed by washing, and the remaining RNA was eluted with elution buffer. Finally, total RNA was reverse‐transcribed to complementary DNA (cDNA) and detected by qRT‐PCR.

RB1-null C33A and RB1-WT HEK293 cells (10⁷ cells per condition) and SH-SY5Y neuroblastoma cells (20⁷ cells per condition) were cultured in 15-cm dishes, transfected with the indicated expression plasmids, and lysed with ice-cold NP-40 buffer supplemented with protease and phosphatase inhibitors (Roche). All cells were treated with dithiobis succinimidyl propionate (Thermo Fisher Scientific) directly on plastic. Cell lysates were passed 20 times through a 25-gauge needle, incubated on ice for 1 hour, pelleted by centrifugation at 12,000 relative centrifugal force (RCF) for 10 min, precleared by 30 min coincubation with magnetic beads (Life Technologies) at 4°C, and incubated overnight at 4°C with 5 μg of the indicated antibodies per 1 mg of quantified protein lysates. Antibody-protein conjugates were captured by incubation with protein G Dynabeads for 1 hour, washed with ice-cold coimmunoprecipitation buffer, and eluted by boiling in SDS running buffer. Total precleared lysates were used as positive control. Antibodies used for these experiments are listed in data S4.

Biotin-labeled probes against circSCAP were designed and synthesized by Sangon Biotech (Shanghai, China). The interaction between circSCAP and miR-7 in NSCLC cells was verified by the RNA pull-down assay. In brief, the cells were transfected with bio-circSCAP by Lipofectamine 3000 (Thermo Fisher Scientific, USA) following the supplier’s procedures. After transfection for 48 h, the cells were harvested and lysed. The cell lysates were incubated with magnetic beads (Life Technologies, USA). After three washes with 1 × PBS, the expression level of miR-7 was determined by qRT‐PCR (Hong et al. 2020 (link)).