Notebooks for reproducing all figures and analysis in the Whole Animal Multiplexed Single-Cell RNA-Seq Reveals Transcriptional Shifts Across Clytia Medusae Cell Types preprint.
All notebooks, saved as .ipynb's, can be run from Google Colab. Colab links are included in every notebook.
Walkthrough of notebooks here. Make your own gene expression plots here.
All saved/processed data used for analysis is streamed to the notebooks from CaltechData.
Data Files:
Raw Counts for Starved/Control Animals
Log-normalized Counts with HVGs for Starved/Control Animals
Fastq Processing Notebooks:
kallisto Fastq Processing for All Experiments
Interactive Browser:
UCSC Genome Browser Page with Interactive Browser
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Preprocessing - All notebooks for ClickTag and cDNA Library Demultiplexing
a) ClickTagDemultiplexing
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cellRangerClickTagCounts.ipynb
- Demultiplexing of ClickTag fastqs for starvation experiment from Cell Ranger bam files
- Input: Cell Ranger bam files of ClickTags
- Output: Count matrices for ClickTags
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kallistobusStarvClickTags.ipynb
- Demultiplexing of ClickTag fastqs for starvation experiment with kallisto-bustools workflow
- Input: Raw ClickTag fastqs
- Output: Count matrices for ClickTags (compared to previour count matrices)
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kallistobusStimClickTags.ipynb
- Demultiplexing of ClickTag fastqs for stimulation experiment with kallisto-bustools workflow
- Input: Raw ClickTag fastqs
- Output: Count matrices for ClickTags and filtered cell barcodes
b) cDNAFiltering
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filterStarvCells_ClickTags.ipynb
- Filtering of cell barcodes from cellRangerClickTagCounts ClickTag counts
- Input: ClickTag count matrices
- Output: Filtered cell barcodes and condition labels
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kallistoBusRuns_StarvAndStim.ipynb
- (Gene) Quantification of cDNA fastqs from starvation and stimulation experiments with kallisto-bustools
- Input: Raw cDNA fastqs
- Output: Anndata objects of cell x gene count matrices for each experiment
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CellAtlasAnalysis - All notebooks for Clustering and Perturbation Response Analysis for Starvation Experiment
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cellRangerClustering_Starvation.ipynb
- Initial clustering of cells from starvation experiment from raw Cell Ranger (cDNA) matrices
- Input: Cell Ranger matrix
- Output: Anndata object of cell x gene count matrices for starvation experiment with clusters and highly variable genes
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starvation_Analysis.ipynb
- Clustering of cells from starvation experiment from kallisto-processed matrices (compared to cellRangerClustering_Starvation output)
- Input: kallisto anndata output
- Output: Anndata object of cell x gene count matrices for starvation experiment with clusters and highly variable genes and analysis of perturbation response
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deSeq2Analysis_StarvationResponse.ipynb
- DeSeq2 analysis for extracting perturbed genes from starved cells
- Input: Clustered anndata object from starvation_Analysis
- Output: Genes that are differentially expressed under starvation ('perturbed' genes)
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neuronSubpop_Analysis.ipynb
- Sub-clustering of neural cell types
- Input: Clustered anndata object from starvation_Analysis
- Output: Anndata object of cell x gene count matrices for neural cells with clusters and marker genes for subpopulations
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fullTrajAnalysis.ipynb
- Pseudotime analysis of neural and nematocyte cell types from the i-cell population
- Input: Clustered anndata object from starvation_Analysis
- Output: Ranking of genes contributing to pseudotime trajectories of neural and nematocyte cell types
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StimulationAnalysis - Clustering and Perturbation Response Analysis for Stimulation Experiment
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stimulation_Analysis.ipynb
- Clustering of cells from stimulation experiment from kallisto-processed matrices
- Input: kallisto anndata output
- Output: Anndata object of cell x gene count matrices for stimulation experiment with clusters and highly variable genes and analysis of perturbation response
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deSeq2Analysis_StimulationResponse.ipynb
- DeSeq2 analysis for extracting perturbed genes from stimulated cells
- Input: Clustered anndata object from stimulation_Analysis
- Output: Genes that are differentially expressed under stimulation (DI/KCl) ('perturbed' genes)
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ComparativeDistanceAnalysis - All Distance-based Analysis for Cell Type/State Delineation and Cross-Experiment Batch Effects
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allDistanceCalculations.ipynb
- Calculations for inter- and intra- cluster distances in starvation and stimulation experiments
- Input: Clustered anndata objects from stimulation_Analysis and starvation_Analysis
- Output: Merged dataset from both experiments and distance-based analysis
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---------- For (User) Gene Searching and Plotting ----------
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SearchAndPlotInteractive - Notebooks for Exploring and Searching Cell Atlas
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MARIMBAAnnosAnalysis.ipynb
- Quantification of raw starvation cDNA fastqs with kallisto-bustools workflow and MARIMBA annotation
- Input: Raw cDNA starvation fastqs
- Output: Anndata object of cell x gene count matrices for starvation experiment with clusters and highly variable genes (compared to starvation_Analysis)
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MARIMBAAtlasSearchAndPlot.ipynb
- Plotting of genes of interest on MARIMBA-quantified anndata object
- Input: Clustered Anndata object from MARIMBAAnnosAnalysis, genes of interest
- Output: Gene expression profiles on cell atlas
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cellAtlasSearchAndPlot.ipynb
- Plotting of genes of interest on Trinity-quantified anndata object (used for all non-Marimba notebooks/main paper analysis)
- Input: Clustered anndata object from stimulation_Analysis, genes of interest
- Output: Gene expression profiles on cell atlas and neuron subpopulations
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- Tara Chari
Several of the pre-processing workflows and initial analyses for the project were first implemented by Jase Gehring.