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Cross linking mass spectrometry of Exo84 pulldown in Sec9 and Sec18 depletion mutants

ABSTRACT: Co-Immunoprecipitation with Crosslinking Using DSS for Exo84 Subunit Sample Preparation: Yeast strains expressing Exo84-3xGFP and FRB-tagged Sec18 or Sec9 were cultured in YPD at 30°C until OD600 0.8-1. Cells were harvested, washed with resuspension buffer (50 mM HEPES, 150 mM NaCl, protease inhibitors), and frozen in liquid nitrogen. Lysis was performed using a Freezer/Mill, followed by clarification via centrifugation. Crosslinking with DSS: Lysates were crosslinked with DSS (0.5 mM, 1h, RT) and quenched with glycine (100 mM, 15 min). Supernatants were clarified by centrifugation. Immunoprecipitation: GFP-Trap beads were used to capture Exo84 complexes. Lysates were pre-cleared with blocking beads (1h, 4°C) before incubation with GFP-Trap beads (overnight, 4°C). Beads were washed with a stringent buffer (2M NaCl, 8M urea, 1% Triton X-100) and PBS. Mass Spectrometry Sample Preparation: Immunoprecipitated complexes were reduced (DTT), alkylated (IAA), digested with trypsin (37°C, 8h), and desalted before LC-MS/MS. LC-MS/MS Analysis: Samples were analyzed on an Orbitrap Fusion Lumos coupled to an EASY-nLC 1200 with a 50 cm C18 column. Peptides were separated with a gradient from 5% to 40% acetonitrile over 60 min. MS1 scans were acquired at 120,000 resolution, and the Top Speed method was used for data-dependent MS2 acquisition with HCD fragmentation.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast) Saccharomyces Cerevisiae

SUBMITTER: Oriol Gallego

LAB HEAD: Oriol Gallego Moli

PROVIDER: PXD061766 | Pride | 2026-01-21

REPOSITORIES: Pride

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			Action	DRS
	2022LB012_LABE_001_01_45pto.msf	Msf
	2022LB012_LABE_001_01_45pto.raw	Raw
	2022LB012_LABE_002_01_45pto.msf	Msf
	2022LB012_LABE_002_01_45pto.raw	Raw
	2022LB012_LABE_003_01_45pto.msf	Msf

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Project description:Glioblastoma stem cells (TS-543) were harvested and cross-linked with 1% formaldehyde for 10 mins at room temperature. The cells were lysed using SDS Lysis buffer for ChIP (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8). The lysate was then sonicated for 25 cycles at 30% amplitude (15 s ON and 45 s OFF). The sonicated samples were then diluted in ChIP dilution buffer (0.01% SDS, 1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8, 167 mM NaCl) and used for the immunoprecipitation with H3K27ac (Abcam, Ab4729), H2AZ (Active motive, cat#39113) and H3K27ac (Abcam cat#ab8580). After an over-night incubation with antibody, the bound DNA was washed sequentially with low salt wash buffer (0.1% SDS, 1% Triton X 100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 150 mM NaCl), high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris–HCl pH 8, 500 mM NaCl), LiCl wash buffer (0.25 M LiCl, 1% NP40, 1%deoxycholate, 1 mM EDTA, 10 mM Tris–HCl pH 8) and TE wash buffer (10 mM Tris–HCl pH 8, 1 mM EDTA) to remove non-specific sequences and eluted in the elution buffer (84 mg NaHCO3, 1 ml 10% SDS, 9 ml H2O). Then the samples were reverse cross-linked using NaCl at 65°C overnight. The eluted DNA was purified and used for library preparation. Library preparation was performed as previously described as described in Bowman et al. BMC Genomics 2013. Briefly, end repair with NEBNext End Repair enzyme (NEB, E6050) and clean-up with 2.4x volume AMPure XP beads (Beckman Coulter, A63880). A-tailing with Klenow Fragment (NEB, M0212) and purified with 2.4x volume AMPure XP beads. Adapter ligation reactions contained annealed universal adapter and T4 rapid ligase (NEB, B0202) and clean-up with 1.8x volume AMPure XP beads. Chromatin was amplified using KAPA Real-time Library Amplification Kit (KAPABIOSYSTEMS, KK2701) with universal primer and barcoded primer. Amplified chromatin was purified with QIAquick Gel Extraction Kit (Qiagen) and sequenced paired-end with Illumina NextSeq 500.

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Cross linking mass spectrometry of Exo84 pulldown in Sec9 and Sec18 depletion mutants

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