Riboprobe systems with t7 rna polymerase

To determine the ASH2L binding sites in USP30-AS1, the full length of USP30-AS1, the antisense of USP30-AS1, as well as the specific regions of the lncRNA were synthesized and labeled with biotin using the Biotin RNA Labeling Mix (Roche, Basel, Switzerland) and the Riboprobe Systems with T7 RNA polymerase (Promega, Madison, WI, USA). For the RNA pull-down assay, 3 µg of the purified biotin-labeled RNA probes were incubated with chondrocyte lysates for 4 h at room temperature and subsequently with streptavidin magnetic beads (Thermo Fisher Scientific) overnight at 4 °C. The bound proteins in the pull-down product were analyzed by western blot using ASH2L antibody.

Biotin-labeled full-length LINC00669 and antisense LINC00669 were synthesized in vitro using Biotin RNA Labeling Mix (Roche) and the Riboprobe Systems with T7 RNA polymerase (Promega). After DNase I treatment, the RNA probes were purified with RNeasy Mini Kit (QIAGEN). For each pulldown assay, 30 μg of biotin-labeled RNA probes were incubated with CNE-2 cell lysates at room temperature for 4 h followed by adding pulling down the binding protein partner with streptavidin magnetic beads (TermoFisher, USA) at 4 °C overnight. The proteins were then separated by electrophoresis and visualized with sliver staining. The unique bands pulled down by sense LINC00669 were subject to mass spectrometry and retrieved in human proteomic library.

Full-length LOC101928120 was synthesized and labelled with biotin using Biotin RNA Labelling Mix (Roche, Basel, Switzerland) and the Riboprobe Systems with T7 RNA polymerase (Promega, Madison, WI, USA). For the RNA pull-down assay, 3 µg of the purified biotin-labelled RNA probes were incubated with chondrocyte lysates for 4 h at room temperature and subsequently with streptavidin magnetic beads (Thermo Fisher Scientific) overnight at 4 °C. The proteins pulled down by LOC101928120 were further analyzed by western blot to detect the level of HDAC1 protein.
To determine the HDAC1 binding sites in LOC101928120, full-length sense and antisense LOC101928120, and specific regions of the lncRNA were synthesized and labelled with biotin using the Biotin RNA Labelling Mix. Cell lysates were incubated with the RNA probes and the standard pull-down procedure was performed. The bound proteins in the pull-down product were analyzed by western blot using HDAC1 antibody.