Halotev protease

The coding region of human USP2A (accession number NM_004205) without the stop codon was cloned into pcDNA3.2/V5-DEST (Thermo Fisher Scientific) using BP clonase II (Thermo Fisher Scientific). The resultant plasmid encoded C-terminal V5-tagged USP2A. Halo-tagged Oct-1 and Oct-2 expression plasmids were purchased from Promega (Madison, WI). The HA-tagged ubiquitin plasmid was purchased from Addgene (Cambridge, MA). The USP2A, or control pcDNA3.2/V5-DEST plasmid, plasmids encoding Halo-tagged Oct-1 or Oct-2 and HA-tagged ubiquitin were transfected into HEK293FT cells using Lipofectamine 2000 reagent (Thermo Fisher Scientific). The pull-down and elution of Oct proteins were performed using Magne HaloTag beads (Promega) and HaloTEV protease (Promega) as per the manufacturer's instructions. For detection of Western blot bands, corresponding to K48- and K63-linked ubiquitin chains, Oct proteins, HA-tagged ubiquitin, and V5-tagged USP2, 1000-fold-diluted antibodies against polyubiquitin chains formed by K48 residues (ab140601; Abcam) and K63 residues (ab179434; Abcam), Oct-1 (A301-717A; Bethyl Laboratories), Oct-2 (ab179808; Abcam), HA-tag (#3724; CST), and V5-tag (A190-120F; Bethyl Laboratories) were used as the primary antibodies.

The MARCC resins were prepared by incubation of saturating amounts (more than 10 nmole) of purified recombinant (HQ)₅-HaloTag proteins with 200 μl HaloLink resin slurry (Promega) in MARCC buffer (30 mM HEPES, 150 mM NaCl, pH 7.4, 0.01% NP-40, 10% glycerol) at 4°C for 1 hour. (HQ)₅-HaloTag protein alone was included as a negative control. Excessive proteins were removed by three brief washes with MARCC buffer. Chromatin capture was achieved by incubating 1 nmole native mononucleosomes or reconstituted nucleosomes with MARCC resins under constant rotation at 4°C overnight. Bound chromatin was further washed with MARCC buffer and eluted by Halo-TEV protease (Promega) cleavage in the presence of 150 μl 1 mM Tris-HCl, pH 7.4 at room temperature for 2 hours. Eluted chromatin was combined with another 150 μl resin-recovered chromatin and could be used for downstream analysis. For small-scale enrichment, 100 pmole nucleosomes and 20 μl MARCC resin were used.

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.