DOI: 10.25345/c5222rj58
title: MassIVE MSV000096938 - MB135-iDUX4ca HSATII RNP RICK LC/MS
title: RICK was performed as previously described (Bao et al., 2018). Briefly, cells were cultured in 15cm plates and 0.1mM 5-ethynyl uridine (ThermoFisher Scientific) was incubated with cells after dox-induction for 16-hrs followed by a washout and then fixed at 48-hrs. After washing 3x with 1X PBS, the plates were placed on ice and irradiated with 0.15 J/cm2 UV light at 254 nm (Stratalinker). Cells were then fixed with 90% ethanol for 30 min, washed 3x with 1X PBS, and permeabilized with 0.5% Triton X-100 in 1X PBS for 15 min. Permeabilized cells were incubated with 15 mL of click reaction buffer (0.25M biotin-azide (Click Chemistry Tools, cat# 1265-25), 0.5M CuSO4, 0.25M THPTA, 0.1M sodium L-ascorbate in 1X PBS) for 30 minutes at RT. Reaction was quenched 3x in 0.5% Triton X-100 in 1X PBS with 2mM EDTA for 3 minutes at RT. Cells were then lysed in lysis buffer (20 mM Tris-HCl, pH 7.5, 500 mM LiCl, 1 mM EDTA pH 8.0, 0.5% lithium-dodecylsulfate (LiDS), and 5 mM DTT) supplemented with Pierce Protease Inhibitors EDTA-free (PIA32955) and Pierce Phosphatase Inhibitors (PIA32957) and RNase inhibitor (TaKaRa, cat# 2313A), and harvested by scraping. Samples were sonicated in a Biorupter (Diagenode) for 15 min on low, 30 sec on/ 30 sec off to aid in lysis. Samples were centrifuged at 12,000 rcf for 10 min at 4C and 5% of the lysate was taken out as an input control. Complexes containing different RNA species, and their associated proteins, were isolated with streptavidin-conjugated magnetic beads (100 ul beads for each plate; Dynabeads MyOne Streptavidin C1, Thermo Fisher Scientific, cat# 65602). After incubation with the lysates overnight under continuous rotation at 4C, the beads were isolated on a magnetic stand and washed using lysis buffer, buffer 1 (20 mM Tris-HCl, pH 7.5, 500 mM LiCl, 1 mM EDTA pH 8.0, 0.1% LiDS, and 5 mM DTT), buffer 2 (20 mM Tris-HCl pH 7.5, 500 mM LiCl, 1 mM EDTA pH 8.0, and 5 mM DTT), and buffer 3 (20 mM Tris-HCl pH 7.5, 200 mM LiCl, 1 mM EDTA pH 8.0, and 5 mM DTT) for two times each under rotation (for 10 minutes at 4C). At last wash step beads were split to isolate RNA or proteins. Proteins were extracted from the captured complexes using RNase A/T1 mix (ThermoFisher Scientific, cat# EN0551) and Ambion RNase III (ThermoFisher Scientific, cat# AM2290) at 37C for 1 hour at 1,000xg, then boiled for 10 minutes 95C. RNA was isolated in RNA elution buffer (10 mM EDTA, pH 8.0, 95% formamide (ThermoFisher Scientific)) and incubated at 95C for 5 minutes at 1,000xg. Proteinase K was added (20mg/mL, ThermoFisher Scientific, cat# AM2546) and incubated at 55C for 1 hour. RNA was extracted using TRIzol. For LC-MS, eluted protein samples were electrophoresed into a NuPage 4-12% Bis-Tris gel, excised, and processed by the Fred Hutchinson Cancer Research Center Proteomics Core. Samples were reduced, alkylated, digested with trypsin, desalted, and run on the Orbitrap Eclipse Tribid Mass Spectrometer (ThermoFisher Scientific). Proteomics data were analyzed using Proteome Discoverer 2.4 against a UniProt human database that included common contaminants using Sequest HT and Percolator for scoring. Results were filtered to only include protein identifications from high-confidence peptides with a 1% false discovery rate. Proteins that were identified in all replicates from both independent experiments with at least two unique peptide matches and greater than 1.5 difference between sample and control were analyzed further.