Rna immunoprecipitation kit

The Ago2-RIP assay was performed using RNA Immunoprecipitation Kit (Millipore, Germany) according to the manufacturer's protocol. The magnetic beads were incubated with AGO2 antibody or IgG. The immunoprecipitated RNAs were further analyzed by qRT-PCR. Total RNAs served as input control.

The interaction between miR-378b and circZNF609 was analyzed by using RNA Immunoprecipitation kit (Millipore) following the manufacturer’s instructions. In brief, U251 and LN229 cells transfected with miR-378b mimics or the NC were collected and lysed. The lysates were added with beads conjugated with Ago2 or IgG antibodies. Then the beads were washed, and subjected to western blotting and qPRC for detection of circZNF609.

RNA Immunoprecipitation Kit (17‐704, Millipore, Burlington, MA, USA) was used for RIP assays. Briefly, trophoblast cell lysates were incubated with magnetic beads conjugated with antibodies of IgG (1:50, anti‐IgG), γ‐H2AX (1:50, anti‐γ‐H2AX), or BRCA1 (1:50, anti‐BRCA1) overnight at 4 °C. RNAs enriched by proteins on beads were extracted and analyzed by RT‐qPCR. For RIP‐Rnase assays, trophoblast cell lysates were treated with 1 U µL⁻¹ RNase T1 to degrade the unbound regions of lnc‐HZ10 before RIP assays. Then, the fragments of lnc‐HZ10 bound with protein were eluted and analyzed as described above. The primer sequences are shown in Table S4, Supporting Information.

LPS, Claycomb Medium, fetal bovine serum (FBS), norepinephrine, and l-glutamine were purchased from Sigma-Aldrich (USA). Dulbecco’s modified Eagle medium (DMEM) was from Thermo Fisher Scientific (USA). QKI encoding vector and adenoviruses were purchased from Vigene Biosciences (China). JetPRIME and JetPEI-Macrophage transfection reagents were from Polyplus Transfection (France). Antibodies against CD68, inducible nitric oxide (iNOS), arginase-1 (Arg-1), QKI, and IL-1β were obtained from Abcam (USA). Antibody against CACNA1C was purchased from Santa Cruz (USA). RNA immunoprecipitation kit was from Millipore (USA). Chromatin immunoprecipitation (ChIP) kit was obtained from CST (USA). Clodronate Liposomes (CL) was from http://clodronateliposomes.com (The Netherlands). Promoter-Binding TF Profiling Plate Array kit was purchased from Signosis (USA).

The RIP experiment was performed according to the instruction manual of RNA Immunoprecipitation Kit (SIGMA). The general experimental steps are as follows: EIF4G2 antibody (abcam, England) or mouse IgG were incubated with magnetic beads for 2 h. Cell lysates were then cultured overnight together with magnetic beads, the proteins in the RNA-protein complex were washed, digested, and the RNA was purified for real-time PCR detection.

According to the instructions of RNA Immunoprecipitation Kit (Sigma-Aldrich), H9c2 cells were lysed and then the cell lysates were incubated with magnetic beads conjugated with antibodies against IgG (anti-IgG) or Ago2 (anti-Ago2) overnight at 4 ℃. Then, qRT-PCR was used to determine the enrichment of circRbms1 and miR-742-3p.