DOI: 10.25345/c5ns0m938
title: MassIVE MSV000096941 - MB135 iDUX4ca EU labeled ChIRP Mass Spec
title: ChIRP was performed as previously described (Chu et al., 2012) with modifications. 6-10 15 cm plates of cells were used per ChIRP-MS experiment. Cells are cross-linked in 2% paraformaldehyde for 15 minutes at RT, followed by 0.125 M glycine quenching for 5 minutes, scraped and pellet at 650xg for 5 minutes at 4C. Cells were then lysed in lysis buffer (50 mM Tris-HCl, pH 7.0, 10 mM EDTA, pH 8.0, 1% SDS supplemented with fresh 0.5 mM AEBSF (ThermoFisher Scientific, cat# BP635-500), Pierce Protease Inhibitors EDTA-free (PIA32955) and Pierce Phosphatase Inhibitors (PIA32957) and RNase inhibitor (TaKaRa, cat# 2313A)) for 10 minutes on ice. Samples were sonicated in a Biorupter (Diagenode) for 15 minutes on high, 30 sec on/ 30 sec off pulse intervals for 4-cycles. Samples were centrifuged at 16,000xg for 10 minutes at 4C and 5% of the lysate was taken out as an input control. Lysates were pre-cleared with beads (Invitrogen Dynabeads M-280 Streptavidin, cat# 11205D) at 37C for 30 minutes at 800rpm. Hybridization was performed at 37C for 4 hours shaking at 800rpm using biotin-conjugated probes (HSATII probe: 5`-ATTCCATTCAGATTCCATTCGATC-3BioTEG-3`, Control probe: 5`-GTCCCGTTAGCTCAGGTGGTAGAGCAC-3BioTEG-3` or 5`-TGCTGATGAAGCAGAACAAC-3BioTEG-3`) in hybridization buffer (750 mM NaCl, 1% SDS, 50 mM Tris-HCl, pH 7.0, 1 mM EDTA, 15% formamide (Sigma-Aldrich) and supplemented with fresh 0.5 mM AEBSF (ThermoFisher Scientific, cat# BP635-500), Pierce Protease Inhibitors EDTA-free (PIA32955) and Pierce Phosphatase Inhibitors (PIA32957) and RNase inhibitor (TaKaRa, cat# 2313A)). Streptavidin magnetic beads (Invitrogen Dynabeads M-280 Streptavidin, cat# 11205D) were added (100 uL per 100 pmole of probes used) and incubated at 37C for 30 minutes at 800rpm. Beads were washed 3x in pre-warmed (37C) wash buffer (2X SSC (Invitrogen), 0.5% SDS and supplemented with Pierce Protease Inhibitors EDTA-free (PIA32955) and Pierce Phosphatase Inhibitors (PIA32957) and RNase inhibitor (TaKaRa, cat# 2313A) and 1 mM DTT) for 5 minutes at 37C at 800rpm. 10% of beads were removed for RNA isolation and 90% of remaining beads were used for protein isolation. 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 and validation of HSATII enrichment was performed using RT-qPCR. Protein was isolated by boiling beads for 30 minutes at 95C in 2X NuPage LDS sample buffer (ThermoFisher Scientific, cat#NP0007). Protein samples were using in immunoblotting or for LC-MS. 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 greater than ten unique peptide matches and greater than 1.5 difference between sample and control were analyzed further.