RNA antisense purification with mass spectrometry (RAP-MS)

RAP-MS was performed as per the protocol described [³⁷]. In brief, the cells were cross-linked in a Spectrolinker at 254 nm wavelength with 0.8 J/cm². Cell lysis was performed using a combination of sonication and DNase treatment. A total of 8 million cells, 1 µg of probe and 200 µl of streptavidin-coated magnetic beads were used for each RAP experiment. The mix of lysate and probe was incubated at 67°C for 2 hours. Beads with lysate were incubated at 67°C for 30 minutes. After 4 washes in 1X hybridization buffer, beads were resuspended in 1 ml of benzonase elution buffer, and 125 U of benzonase non-specific nuclease was added. Samples were incubated at 37°C for 2 hours, and beads were removed. To precipitate proteins, a 10% final concentration of trichloroacetic acid (TCA) was added. After overnight incubation at 4°C, samples were centrifuged at 16,000 G for 30 minutes. The supernatant was removed and replaced with 1 ml of cold acetone. Then, centrifugation at 16,000 G for 15 minutes was performed. The MS/MS data were analysed using Mascot (Matrix Science, London, UK). Mascot was set up to search against the UniProt human database using the following criteria: peptide mass tolerance ± 5 ppm, fragment mass tolerance ± 0.08 Da and maximum missed cleavages: 1. Positive peptide identification was determined if they could be established at greater than 95% probability as specified.