single-cell-histone-ptm (sc-hPTM)

Repository for processed data and scripts used in the manuscript: Mass Spectrometry-based Profiling of Single-cell Histone Post-translational Modifications to Dissect Chromatin Heterogeneity

Data Structure

1. data - Processed bulk and single-cell data

Bulk Data

• Dele-Oni et al 2021 - DOI: 10.1038/s41597-021-01008-4. Global chromatin profiling assay level 3 processed Normalized and QC'ed Numerical Data (QCNORM) retrieved from: PanoramaWeb.
• Sidoli et al 2015 - DOIs: 10.1021/acs.analchem.5b00072, 10.1021/acs.analchem.5b03009. Processed global chromatin profiling assay data taken from supplementary tables.

Single-cell Data

• Sample table with metadata for injections.
• Intensities
  • *_cells.csv - Processed global chromatin profiling assay retention time and intensity values (MS1 and MS2 levels) for cell injections.
  • *_hist_stand.csv - Processed global chromatin profiling assay retention time and intensity values (MS1 and MS2 levels) for histone standard injections.
  • *_prop_efficiency.csv - Processed derivatization peptides retention time and intensity values (MS1 and MS2 levels).

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2. derivatization-efficiency

• Script for calculating derivatization efficiency of single-cells from batches 1-5.

3. calibration-curves

Cells

• Script for plotting peptidoform titration curves and calculating correlation coefficients of cell injections.

Histone-standards

• Script for plotting peptidoform titration curves and calculating correlation coefficients of histone standard injections.

4. normalization

Auto

• Script for plotting quality control such as intensity distribution and background noise, filtering out low-quality peptidoforms and cells, peptide ratio normalization, batch correction, and data visualization.
  • Used to process cells for benchmarking experiments from batches 1-3.

Auto+NABUT

• Same as the auto script but used for processing batches 1-5.

5. sc-vs-bulk

• Script for comparing pseudobulk single-cell data (batches 1-3) with 100-cell bulk or previous bulk histone studies.

6. technical-variability

• Script for calculating variation across histone standard technical replicates and technical replicates from previous bulk studies.

7. biological-variability

Auto

• Script for calculating cell-to-cell variation at the peptidoform level while accounting for technical variation (measured by histone standards) using single-cell data from batches 1-3.

Auto+NABUT

• Script for calculating cell-to-cell variation at the peptidoform level, cell level, and performing hPTM covariance analysis using single-cell data from batches 1-5.

8. cell-classification

• Script for training a KNN classifier on labeled single-cell data and evaluating it on a test set.
• Re-trains the KNN classifier on all labeled single-cell data to classify unlabeled single-cell data (batches 1-5).

9. differential-abundance

With-mixed

• Differential abundance analysis of bulk and single-cell data, including unlabeled "mixed" cells classified using a KNN classifier.
• Results visualized using volcano plots and heatmaps.
• Control and treatment groups further sub-clustered and visualized.

Without-mixed

• Same differential abundance analysis as above but excludes unlabeled "mixed" cells.
• Includes correlation of results with the "with-mixed" analysis.

10. network-analysis

• Cytoscape analysis file and tables for control and treatment groups containing hPTM-hPTM correlation values and their significance (derived from 7-biological-variability).

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Additional Files

cellenONE_runs

• Run files used with the cellenONE instrument for executing the sc-hPTM sample preparation method.

functions.R

• Contains all functions used throughout scripts in the analysis pipeline.