Biotin rna labeling mix kit

RNA pull-down assays were performed using biotinylated WT or Mut S2 lncRNA-HEIH. Briefly, the biotinylated lncRNA-HEIH S2 RNA was transcribed with a Biotin RNA Labeling Mix Kit (Roche, #11685597910) and T7 RNA polymerase (TaKaRa, #2540 A). Three pmole of transcribed RNA was applied for further pull-down assays. Meanwhile, the HEK293T cell lysates that were transfected with GST-MYC-UPF1 were purified with GST-antibody conjugated beads (GE Healthcare, #17-0756) in the presence of a nuclease mixture (DNase and RNase). One pmole of GST-MYC-UPF1 was incubated with three pmoles of in vitro transcribed RNA for 2 h at 4 °C. Afterward, 40 μl of streptavidin-conjugated magnetic beads (NEB, #S1420S) was added for another hour of incubation at room temperature. The beads were washed ten times and boiled with 1×SDS loading buffer for 10 min at 95 °C. The RNA-bound proteins and RNA were analyzed by WB and RT-sqPCR, respectively.
To overexpress RNA in Huh7 cells, RNA was transcribed in vitro. LncRNA-HEIH and luciferase in pcDNA3.1 were transcribed by T7 polymerase (TaKaRa, #2540 A) in the presence of methylpseudouridine (Jena bioscience, #NU-890S) to evade the innate immune system.

The pcDNA3.1(+) pDRAIR and pcDNA3.1(–) expressing DRAIR anti-sense (pDRAIR-AS, Supplemental Figure 3C) were linearized to prepare biotinylated DRAIR and DRAIR-AS probes, respectively, by in vitro transcription with T7-RNA polymerase using Biotin RNA Labeling Mix kit (Roche). Nuclear extracts from THP1 cells (1 mg protein) were incubated with 1 μg of biotinylated probes, and RNA pulldown assays were performed as described (22 (link)). Proteins eluted from biotinylated RNA-protein complexes were subjected to immunoblotting with G9a antibody (1:1000). Protein bands were detected using Enhanced Chemiluminescence kit (Perkin Elmer).

The biotin‐labelled RNA probe for circ‐CTNNB1 was in vitro transcribed using the Biotin RNA Labeling Mix kit (Roche) and T7 RNA polymerase, as described in our previous study.²³ RNA pull‐down assay was performed at room temperature, and the biotinylated proteins were detected by mass spectrometry (MS) at the Wuhan Institute of Biotechnology (Wuhan, China).

Expression vectors for full-length DIAPH2-AS1 and its truncated fragments were synthesized and transcribed using the T7 RNA polymerase (Promega, Madison, Wisconsin, USA) in vitro. After in vitro transcription, DIAPH2-AS1 and truncated fragments were biotinylated using a Biotin RNA Labeling Mix kit (Roche, Basel, Switzerland, USA). Then the pulldown assay was conducted with a Pierce Magnetic RNA-Protein Pull-Down Kit (Thermo Fisher Scientific, Waltham, MA, USA). Biotin-labeled RNA was incubated with streptavidin magnetic beads at room temperature for 1 h. Total cell lysates were prepared freshly and added Protease/Phosphatase Inhibitor Cocktail and RNase inhibitors in each step of the reactions. Probes-beads complexes were incubated with cell lysates at 4 °C for 6 h. After thoroughly washing to remove unbound proteins, RNA-protein binding complexes were added to SDS buffer and boiled. Subsequently, eluted proteins were observed by silver staining and detected using western blot and Mass Spectrometry analysis.

Biotin-labeled oligonucleotide probes (Additional file 1: Table S4) targeting junction sites of circRNAs were synthesized (Invitrogen). Using Biotin RNA Labeling Mix kit (Roche, Indianapolis, IN, USA) and T7 RNA polymerase, the biotin-labeled RNA probes for circRNAs were in vitro transcribed as previously described [15 (link), 17 (link)]. RNA pull-down assay was performed at room temperature, and retrieved proteins were detected by mass spectrometry analysis at Wuhan Institute of Biotechnology (Wuhan, China).