双虚拟敲除 CellOracle + scTenifoldKnk 全流程复现（二）

TL;DR - Part two of a WeChat tutorial series reproducing a 2026 Cell Prolif paper that used dual virtual knockout (CellOracle + scTenifoldKnk) to identify STAT3 as a dentinogenesis regulator; this installment covers the single-cell QC and cell-type annotation steps on GSE146123 in R/Seurat.

• QC thresholds were chosen after an AI-assisted literature scan of dental pulp/odontogenic scRNA-seq papers (MT% cutoffs ranging 5%–40% across Pang 2023, Yang 2024, Zhang 2026, Liu 2026); the author settled on nFeature_RNA 200–6000, nCount_RNA 3–30000, percent_mito <10%, percent_hb <1%.
• Standard Seurat pipeline: LogNormalize (scale factor 1e4), 2000 variable features, ScaleData, PCA, then Harmony integration on orig.ident to correct sample batch effects, with UMAP/neighbors on dims 1:20.
• Clustering resolution was swept over 0.05–1 and inspected with clustree; resolution 0.5 was used for the working annotation.
• Annotation targets the original paper's 9 major populations (endothelial 21.2%, mesenchymal 20.7%, perivascular 16.9%, glial, peri-odontoblastic layer, pulp, preodontoblasts, epithelial, immune), with marker-based distinctions noted (mesenchymal: COL1A1/DCN/LUM vs. pulp: VIM/BGN/POSTN).
• Note: content is truncated mid-annotation, and the actual CellOracle/scTenifoldKnk perturbation steps are deferred to later installments.