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Phosphoproteomics analysis of HCT-116 cells treated with tumor necrosis factor (TNF)

R/Quarto analysis accompanying:

LUBAC PUB domain interactions restrict Met1-linked ubiquitination to prevent embryonic lethality and immune pathology in mice

This repository contains the phosphoproteomics analysis of: HOIP KO HCT-116 human colon carcinoma cells, reconstituted with HOIP WT or with HOIP(N102D), following TNF stimulation for 5 and 15 minutes.

Data

The phosphoproteomics data are deposited in PRIDE.

PRIDE accession: PXD060649

The analysis starts from:

20241126_Report_PTM_pivot_GF (Pivot).tsv

Place this file in the data/ folder before running the analysis.

Analysis

The complete analysis is contained in analysis.qmd and includes:

  • data filtering and quality control
  • log2 transformation and median centering
  • differential phosphorylation analysis with limma
  • regulated phosphosite summaries and overlap analysis
  • phosphosite heatmaps
  • KEGG TNF and NF-kB pathway heatmap

Helper functions used by the analysis are stored in R/functions.R.

Running the analysis

Open TNF_project.Rproj in RStudio and render analysis.qmd.

Required R packages are listed at the beginning of the analysis.

Input data and generated outputs are kept locally and are excluded from the Git repository.

Repository structure

analysis.qmd        Main phosphoproteomics analysis
R/functions.R       Helper functions
TNF_project.Rproj   RStudio project
data/               Input data (not tracked by Git)
outputs/            Generated tables and figures (not tracked by Git)