Streptavidin magnetic beads

To enrich RNAs directly interacted with Circ_0000915, three specific antisense biotin-labeled probes against Circ_0000915 were incubated with HemSCs cell lysates. Their corresponding sense biotin-labeled probes were used as the negative control. Streptavidin magnetic beads (Invitrogen) were used to absorb biotin-labeled probes-enriched fractions 4 h after incubation. Beads were washed for RNA extraction and the extracted RNA was subsequently subjected to qRT-PCR analysis. Biotin-labeled oligonucleotides used here were all listed in Additional file 1: Table S1.
For biotin miRNA pull-down assay, the detailed protocol was described previously [34 ]. Biotinylated RNAs of miR-890 (Biotin-miR-890) and negative control (Biotin-miR-NC) were purchased from GenePharma (Shanghai, China).

The biotin-labeled miR-483-5p probe and streptavidin magnetic beads (Invitrogen) were incubated at room temperature and then incubated with TCMK-1 cell lysate or primary mouse renal TEC lysate. Subsequently, the magnetic beads were washed with lysis buffer, and the TIMP2 and MAPK1 bound to the magnetic beads were analyzed by qRT-PCR or the HNRNPA1 protein bound to the magnetic beads was analyzed by western blot.

We used a previously published human naïve B cell phage library with a diversity of 4.1 x 10¹⁰ to identify Fabs against VCBC (23). Fabs were isolated using a phage display panning protocol descirbed in detail by Kim et al, 2011 [24 (link)]. Briefly, purified VCBC was biotinylated using EZ-Link NHS-Chromogenic-Biotin (Pierce) as described previously [24 (link)]. A fully human naïve Fab phage display library was used to identify antibodies against VCBC complex [23 (link),24 (link)]. The panning was accomplished in four rounds using streptavidin magnetic beads (Invitrogen) coated with biotinylated VCBC complex as described previously. After four rounds of selection, Fabs that bind to VCBC were identified by an ELISA screen [41 (link)]. Clones with a positive signal in ELISA were analyzed by BstNI restriction analysis to identify the unique clones. scFvs were generated by linking the heavy and light chain variable regions together with a 24 aminoacids linker (ASSGGSTSGSGKPGSGEGSSGSAR), and then cloning these constructs into pSYN1 or pcDNA4, for E.coli or mamalian expression, respectively.

RNA pull-down assays were performed to prove the interaction between GAS5 and miR-21. Full‐length GAS5 was transcribed in vitro with T7 RNA Pol II (Promega, Madison, WI) and labelled with biotin using Biotin RNA Labeling Mix (Roche, USA). An oligo probe (Bio-NC) was used as the NC. HeLa cells were fixed with formaldehyde to cross-link RNA and then suspended in 1 ml of lysis buffer (20 mM Tris-HCl, 200 mM NaCl, 2.5 mM MgCl₂·6H₂O, 0.05% IGEPAL) containing RNase and protease inhibitors. Three micrograms of biotinylated RNA (Bio-GAS5, Bio-NC) was incubated upside down with Streptavidin Magnetic Beads (#88816, Invitrogen) at room temperature for 2 h. Then, we added 500 μl of cell lysate supernatant to the magnetic beads for another 2 h of incubation at 4 °C. After sequential elution with solution A (100 mM NaOH, 50 mM NaCl) and solution B (100 mM NaCl) three times, the magnetic beads were resuspended in 200 μl of lysis buffer and rotated at room temperature for 2 h to release the formaldehyde cross-linked RNA in the sample. The RNA–RNA complexes were purified using TRIzol reagent (Takara). The miR-21 level in the RNA–RNA complexes was detected by qRT-PCR.

For the detection of miRNAs pulled down by circ-APC, U2932 and TMD8 cell lysates were collected and incubated overnight with a biotinylated control (5′-GCTAAAGTCAAGTCTGAAAAGCAATGATGTTGTCCACTGG-3′) or circ-APC probe (5′-AAAAGCGAAG AATAGCCAGAA-3′) from RiboBio. Next, the lysates were incubated with streptavidin magnetic beads (Invitrogen) at room temperature for three hours to generate bead/circ-APC complexes. The RNA bound by circ-APC was extracted with TRIzol solution, purified with an RNeasy Mini Kit (Qiagen, Dusseldorf, Germany) and analyzed by qRT-PCR to determine the expression of the 12 indicated miRNAs. In addition, the proteins bound by circ-APC were collected in radioimmunoprecipitation assay lysis buffer, and Western blot analysis was performed to assess TET1 expression.
For the detection of miR-888 pulling down circ-APC, biotinylated wild-type or mutant miR-888 mimics were synthesized and transfected into U2932 and TMD8 cells with Lipofectamine 3000 (Invitrogen). The cells were then incubated with the above streptavidin magnetic beads, and circ-APC expression was detected by qRT-PCR.