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An Immunological Synapse Formation Between T Regulatory Cells and Cancer-Associated Fibroblasts Promotes Tumour Development

ABSTRACT: The cells were lysed in a buffer consisting of 4% SDS, 0.1 M DTT, 100 mM Tris/HCl; pH 7.6 and heated for 5 minutes at 99℃. The protein extracts were processed according to theSp3 protocol. The reduced cysteine residues were alkylated in 200 mM iodoacetamide (Acros Organics). 20 μg of beads (1:1 mixture of hydrophilic and hydrophobic SeraMag carboxylate-modified beads; GE Life Sciences) were added to each sample in 50% ethanol. Protein clean-up was performed on a magnetic rack. The beads were washed twice with 80% ethanol followed by one wash with 100% acetonitrile (Fisher Chemical). The beads-captured proteins were digested overnight at 37 ℃ with 0.5 μg trypsin/LysC mix in 25 mM ammonium bicarbonate under vigorous shaking (1200 rpm, Eppendorf Thermomixer). The supernatants were collected and the peptides were purified by a modified Sp3 clean-up protocol and finally solubilized in the mobile phase A (0.1% formic acid in water), and sonicated. Peptide concentration was determined through absorbance measurement at 280 nm using a nanodrop instrument.

INSTRUMENT(S): Q Exactive HF-X

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Skin, Fibroblast

DISEASE(S): Melanoma

SUBMITTER: Martina Samiotaki

LAB HEAD: Panayotis Verginis

PROVIDER: PXD047024 | Pride | 2024-05-08

REPOSITORIES: Pride

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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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An Immunological Synapse Formation Between T Regulatory Cells and Cancer-Associated Fibroblasts Promotes Tumour Development

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