Ribocluster profiler rip assay kit

p62/IMP2-bound RIP was performed according to the RiboCluster Profiler RIP-Assay Kit (Medical & Biological Laboratories CO., LTD. Japan). Briefly, cell lysates were collected from p62/IMP2 positive cells, and then the whole complexes of p62/IMP2-targets from cell lysate were pulled down with p62/IMP2 antibody. The target mRNAs were separated and isolated for subsequent reverse transcription.

Immunoprecipitation of ribonucleoprotein complexes was performed using a RiboCluster Profiler RIP-Assay kit (Medical & Biological Laboratories, Nagoya, Japan) according to the manufacturer's protocol. Briefly, HEK293 cells transiently expressing Halo-tagged KHSRP (KHSRP-Halo) were crosslinked on ice by irradiation with UV light (365 nm) at 150 mJ/cm². Cytoplasmic lysates (250 μg protein) or nuclear extracts (250 μg protein) were incubated for 3 h at 4°C with 50 μL of a 50% (v/v) suspension of HaloLink Resin (Promega, Madison, WI, USA). After the resin was treated with HaloTEV protease (Promega), the IP immunoprecipitated transcripts were quantified by qPCR.

Following the manufacturer’s protocol, RIP assay was performed to extract FMRP-bound mRNAs (n = 3, FMRP-IP; n = 2, negative control (IgG-IP)) from E14.5 WT mice cortex by using RiboCluster Profiler™ RIP-Assay Kit with anti-FMRP human polyclonal antibody, RN016P (Medical and Biological Laboratories Co., Ltd.) and Dynabeads™ Protein beads G/A (Invitrogen™). The quality and quantity of the total RNA were evaluated using the Agilent 2100 Bioanalyzer with RNA 6000 Pico Kit (Agilent). Total RNA concentration greater than 50 ng with an RNA Integrity Number (RIN) value greater than or equal to 7.9 was sequenced.