Rnasin

An RNA pull-down assay was performed to evaluate the direct combination between circLMO1 and miR-4291. Streptavidin magnetic beads (M2420; Solar Bio, Beijing, China) were labeled with biotinylated-miR-4291 (Sangon Biotech, Shanghai, China) at 4°C for 12 h. Approximately 0.5×10⁷ CaSki or C33A cells were lysed with commercial lysis buffer (P0013; Beyotime) supplemented with 40 U/mL RNasin (Tiangen Biotech). The lysate was reacted with RNA probe-labeled streptavidin magnetic beads for 3 h at room temperature. Finally, the circLMO1 content of the eluted complex was determined by qPCR.

RIP was done as documented previously [24 (link)]. Concisely, cells were inoculated with Trypsin (Beyotime) and washed with PBS, then crosslinked in 1% formaldehyde for 10 minutes at RT. After that, the cells were inoculated with 2.5 M glycine for five minutes at RT to block formaldehyde. Next, we re-suspended the cell pellet in RIPA buffer (Beyotime) enriched with RNasin (Tiangen, Beijing, China), protease inhibitor cocktail (Beyotime), and 1 mM PMSF (Beyotime). The cell suspension was briefly sonicated and span for three minutes at 14,000 × g at RT. Normal Rabbit IgG (#2729, CST, Beverly, USA) or TCPTP (#58935, CST) antibodies were pre-bound with Protein G Sepharose beads for 3 hours at RT. Thereafter, Protein G Sepharose beads (Abcam, Cambridge, UK) were employed to pre-clear the supernatant and then introduced to the beads and incubated on a rotating wheel overnight at 4°C. Beads were then rinsed with RIPA buffer. We reversed the crosslinks and Proteinase K (Beyotime) was employed to digest the proteins at 65°C for two hours. After that, RNA was extracted by using TRNzol Universal (Tiangen) and reverse-transcripted by using FastKing-RT SuperMix (Tiangen) as documented in the manual provided by the manufacturer.