Single-Cell RNA-Seq Analysis Report

Executive Summary

Study Overview

Total Cells Analyzed

49266

High-quality cells passing QC filters

Seurat Clusters

Unsupervised clusters (resolution 1.0)

Cell Types Identified

Annotated by marker gene expression

DE Genes (All Cells)

3725

Significant DEGs between conditions (all cells)

Materials and Methods

Experimental Design

Sample ID	Sample Name	Condition
s01	treatment_1	treatment
s02	treatment_2	treatment
s03	control_1	control
s04	control_2	control

Analysis Pipeline

Platform: 10x Genomics Chromium; species: human
Quality Control: Standard QC metrics including nCount_RNA, nFeature_RNA, and percent.mt filtering
Doublet detection using Scrublet prior to QC filtering
Scanpy and Python for data processing and integration
Harmony integration across samples
UMAP dimensionality reduction (dims 1:30)
Graph-based clustering (resolution 1.0)
Cell Type Annotation: Client-provided markers for cell types
Wilcoxon rank-sum test for differential expression
GO and KEGG enrichment via gseapy (Enrichr API)

Cell Type Markers

Cell type markers were provided by the client and used to annotate clusters based on known gene expression patterns.

Cell Type	Marker Genes
CTB	EPCAM,TP63,ELF5,KRT7
SCT	CGA,CGB,ERVFRD-1
EVT	HLA-G,ITGA1,NOTCH2
dNK	KLRD1,NKG7,GNLY
neutrophil	FCGR3B,CSF3R,S100A8
Macrophage	CD68,CSF1R,C1QC
Stromal	IGFBP1,PRL,DKK1
Endothelial	PECAM1,VWF
Perivascular	MCAM,RGS5
Hofbauer	SPP1,APOE

Quality Control

Filtering and Quality Metrics

Cells were first processed using Scrublet to identify and remove doublets, ensuring the analysis reflects individual cellular transcriptomes. Subsequent quality control filtered out low-quality cells with fewer than 500 UMIs, fewer than 200 detected genes, or a mitochondrial gene proportion exceeding 20%.

Sample ID	Sample Name	Condition	nCells (orig.)	Median nCount	Median nFeature	% MT < 20%	% Doublets	nCells (filtered)	% Retained
s01	treatment_1	treatment	13,340	3,856	2,086	99.3%	4.7%	12,399	92.9%
s02	treatment_2	treatment	12,003	3,003	1,695	99.6%	2.7%	11,519	96.0%
s03	control_1	control	12,981	2,735	1,691	99.5%	4.0%	12,332	95.0%
s04	control_2	control	13,305	3,553	1,917	99.9%	1.5%	13,016	97.8%

Cell counts from Cell Ranger; median values and filtering statistics computed per-sample.

QC Violin Plots

QC violin plots after filtering — all samples (nCount_RNA, nFeature_RNA, percent.mt)

QC Scatter Plot: nCount vs nFeature

nCount_RNA vs nFeature_RNA

Integration & Clustering

Integration: Filtered, per-sample Seurat objects were integrated using Harmony (Seurat v5, RunHarmony on PCA embeddings, dims = 1:30) to correct for batch effects across samples.
Clustering: Shared nearest-neighbour graph was built (FindNeighbors, dims = 1:30), followed by Louvain graph-based clustering (FindClusters, resolution = 1.0).

Sample and Condition Distribution After Integration

UMAP colored by sample

UMAP colored by condition

UMAP Clustering

UMAP colored by cluster

Cell Type Annotation

Cell type annotation strategy:

Marker gene identification: Differentially expressed marker genes for each cluster were identified using the Wilcoxon rank-sum test (FindAllMarkers, min.pct = 0.25, logfc.threshold = 0.25).
Annotation: Each cluster was manually annotated by comparing its DE marker genes against a curated reference panel of known cell type markers (provided by the client). Clusters sharing the same DE marker signature were merged under a single cell type label.

Cell Type UMAP

UMAP colored by annotated cell type

Marker Gene Dot Plot — All Cell Types

Dot plot of canonical marker genes across all annotated cell types

Marker Gene Expression per Cell Type

CTB — violin plot by cell type

EVT — violin plot by cell type

Endothelial — violin plot by cell type

Macrophage — violin plot by cell type

Perivascular — violin plot by cell type

SCT — violin plot by cell type

Stromal — violin plot by cell type

dNK — violin plot by cell type

neutrophil — violin plot by cell type

Cell Type Summary

Cell Type	Total Cells	% of Total	Clusters
SCT	9,891	20.1%	0, 8, 9, 22, 24
Perivascular	8,894	18.1%	5, 6, 12, 16
CTB	6,630	13.5%	7, 10, 11, 15
Endothelial	6,585	13.4%	4, 13, 17, 20
Macrophage	5,505	11.2%	3, 14, 21
Stromal	4,952	10.1%	1, 25
EVT	4,366	8.9%	2
neutrophil	1,167	2.4%	19, 23
dNK	857	1.7%	18
Others	419	0.9%	26, 27
Total	49,266	100%

Source: Tables/projectID_all_cell_type_num.csv

Cell Counts and Proportions by Sample

	control_1	control_2	treatment_1	treatment_2
CTB	2,041	2,418	1,073	1,098
EVT	998	720	1,319	1,329
Endothelial	2,079	1,315	1,728	1,463
Macrophage	1,064	1,251	2,093	1,097
Perivascular	2,206	1,986	1,996	2,706
SCT	2,557	3,826	1,246	2,262
Stromal	998	647	2,186	1,121
dNK	160	226	372	99
neutrophil	198	478	234	257
Total	12,301	12,867	12,247	11,432

Source: 03.MarkerGene_plot/projectID-celltype-summary.csv

Cell Type Composition Analysis

Composition Plots

Cell type proportion per condition (stacked bar)

Cell type count per condition (stacked bar)

Composition Comparison Between Conditions

Cell type composition bar plot by condition

Cell type number/proportion bar plot by condition

Differential Expression: Between Cell Types

Top Marker Gene Heatmap

Heatmap of top 5 marker genes per cell type

Top Differentially Expressed Genes (between cell types)

Cell Type: CTB (top 5 markers)
Gene	log₂FC	adj. p-value
*IFI6*	7.755	5.38e+00
*PEG10*	9.065	6.28e+00
*PAGE4*	9.056	6.28e+00
*ISM2*	8.495	5.89e+00
*SPINT1*	7.825	5.42e+00

Cell Type: EVT (top 5 markers)
Gene	log₂FC	adj. p-value
F3	6.806	4.72e+00
*COL16A1*	6.193	4.29e+00
*COL1A1*	6.757	4.68e+00
*DCN*	6.021	4.17e+00
*COL5A1*	5.559	3.85e+00

Showing top 5 marker genes for the two most represented cell types. Full DE gene list: 05.DE_cellType/projectID-DE-cellType-genes.csv

Differential Expression: Between Conditions

Differential expression analysis comparing treatment vs control across all cells and per cell type. Comparison groups are defined in groupComparisonSheet.csv.

Comparison: treatment vs control

Overall DE Results

Metric	Value
Total Genes Tested	16,235
Significant DE Genes (p_adj < 0.05)	3725
Upregulated in treatment	2000
Downregulated in treatment	1725

Volcano Plot

Volcano plot — treatment vs control (CTB representative)

Top Differentially Expressed Genes

Most significantly upregulated genes in treatment (top 6)

Gene	log2FC	Adj. p-value
*EGR1*	4.9658	3.44e+00
*ADAMTSL1*	5.0688	3.51e+00
*ECRG4*	4.2737	2.96e+00
*PCDH7*	6.3298	4.39e+00
*DPEP1*	7.9286	5.50e+00
*MXRA5*	4.0048	2.78e+00

Most significantly downregulated genes in treatment (top 6)

Gene	log2FC	Adj. p-value
*CASQ1*	-4.2811	-2.97e+00
*ATP1A4*	-4.1045	-2.85e+00
*ATP2C2*	-5.4475	-3.78e+00
*PCDH11Y*	-6.5382	-4.53e+00
*EIF1AY*	-15.9668	-1.11e+01
*KDM5D*	-14.8209	-1.03e+01

Top DE Genes Feature Plot

Upregulated genes

EGR1 — Control vs Treatment

ADAMTSL1 — Control vs Treatment

ECRG4 — Control vs Treatment

PCDH7 — Control vs Treatment

DPEP1 — Control vs Treatment

MXRA5 — Control vs Treatment

Downregulated genes

KDM5D — Control vs Treatment

EIF1AY — Control vs Treatment

CASQ1 — Control vs Treatment

PCDH11Y — Control vs Treatment

ATP1A4 — Control vs Treatment

ATP2C2 — Control vs Treatment

Top DE Genes Violin Plot

Upregulated genes

EGR1

ADAMTSL1

ECRG4

PCDH7

DPEP1

MXRA5

Downregulated genes

KDM5D

EIF1AY

CASQ1

PCDH11Y

ATP1A4

ATP2C2

DE Summary by Cell Type Subset

Cell Type Subset	Total Sig. DE	Upregulated in treatment	Downregulated in treatment	Result Folder
All	3725	2000	1725	`06.DE_condition/treatment_vs_control/All`
CTB	2118	726	1392	`06.DE_condition/treatment_vs_control/CTB`
EVT	2883	1589	1294	`06.DE_condition/treatment_vs_control/EVT`
Endothelial	1303	597	706	`06.DE_condition/treatment_vs_control/Endothelial`
Macrophage	2422	1056	1366	`06.DE_condition/treatment_vs_control/Macrophage`
Perivascular	1812	1174	638	`06.DE_condition/treatment_vs_control/Perivascular`
SCT	3560	91	3469	`06.DE_condition/treatment_vs_control/SCT`
Stromal	4657	4273	384	`06.DE_condition/treatment_vs_control/Stromal`
dNK	1318	1235	83	`06.DE_condition/treatment_vs_control/dNK`
neutrophil	2204	1909	295	`06.DE_condition/treatment_vs_control/neutrophil`

Number of Differentially Expressed Genes by Cell Type

GO Biological Process Enrichment (Top 10, All DE Genes)

GO Biological Process enrichment — top 10 (all DE genes)

KEGG Pathway Enrichment (Top 10, All DE Genes)

KEGG Pathway enrichment — top 10 (all DE genes)

Conclusions and Biological Interpretation

Summary of Findings

49266 total cells passed quality control filtering and were included in downstream analysis
28 clusters were identified at resolution 1.0
10 cell types were annotated based on known marker gene expression
3725 significant DE genes were identified for treatment vs control (all cells): 2000 upregulated, 1725 downregulated
GO and KEGG pathway analyses were performed for each comparison and cell type subset

Data Files Provided

Folder / File	Contents	Description
`01.QC/`	Quality Control	Per-sample QC plots before and after filtering: violin plots of nCount_RNA, nFeature_RNA, and percent.mt; UMI and gene density plots; mitochondrial fraction distributions; cell number summary CSVs (original and after filtering).
`02.Umap_all/`	UMAP — All Cells	UMAP dimensionality reduction plots for the integrated dataset, colored by Seurat cluster, cell type, sample identity, and condition. Also includes split/per-condition UMAPs.
`03.MarkerGene_plot/`	Marker Gene Plots	Cell type annotation visualizations: marker gene feature plots and violin plots (by cell type and by cluster) for each annotated cell type; combined dot plot and top-marker heatmap across all cell types; cell type count summary CSV.
`04.CellType_proportion_num_compare/`	Cell Type Composition	Cell type composition analysis comparing conditions: stacked bar plots of cell type proportions and counts per sample/condition; grouped comparison bar plots; box plots of proportion variability across samples (per condition); composition CSV tables.
`05.DE_cellType/`	DE — Between Cell Types	Differential expression analysis identifying marker genes that distinguish each cell type from all others (Wilcoxon rank-sum, FindMarkers). Contains: DE gene table CSV, up/down summary plot, top-marker heatmap, and GO/KEGG enrichment results (barplots, dot plots) and GSEA for each cell type.
`06.DE_condition/`	DE — Between Conditions	Differential expression analysis between conditions (treatment vs control). Organized as `treatment_vs_control/{subset}/` where subset is All (all cells combined) or an individual cell type. Each subset folder contains: full and significant DE tables (CSV), volcano plot, heatmaps, dot plots, violin plots, feature plots for top up/down-regulated genes, GO/KEGG enrichment barplots and dot plots, and GSEA enrichment plots and tables.
`Tables/`	Tables & Key Files	Core data objects and supplementary files: annotated Seurat objects at multiple processing stages (.rds); cell-level metadata CSV (barcodes, QC metrics, cluster, cell type, condition); all-markers and top-markers CSV tables; cluster-to-cell-type annotation table; doublet statistics; cell type summary (counts, proportions, clusters); feature plots and violin plots for top DE genes; R analysis scripts.
`cellrangerResult/`	Cell Ranger Output	Raw Cell Ranger multi pipeline output for each sequencing pool (`projectID-pool-1`, `projectID-pool-2`). Each pool folder contains: `per_sample_outs/` with per-sample filtered feature-barcode matrices (used as input to Seurat), and `multi/multiplexing_analysis/` with cell barcode assignment and demultiplexing statistics.
`sampleInfo.csv`	Reference file	Sample manifest: sample IDs and names
`groupComparisonSheet.csv`	Reference file	Comparison group definitions (Treatment vs Control)
`human_placenta_cell_type_markers.txt`	Reference file	Client-provided marker genes used for cell type annotation