Chromium single cell reagent kit

scRNA-seq libraries were prepared using Chromium Single Cell Reagent Kit (10X Genomics). The libraries were then pooled and sequenced on NovaSeq 6000 (Illumina), using NovaSeq Control Software v1.6.0. The raw sequence data were processed using Cell Ranger software (v.3.0.1). The reads were aligned to human genome GRCh38, and a gene count matrix was generated for each sample. The raw count data were then loaded into Seurat package (v3.1.1) for QC, filtering, normalization, Uniform Manifold Approximation and Projection (UMAP) visualization, and clustering (15 (link)). The cells that have mitochondrial genes greater than 10% or have fewer than 200 detected genes were filtered out. A scale factor of 10,000 was used to normalize all the remaining cells. To correct for the batch effect between different samples, and the reciprocal principal component analysis (RPCA) method in the Seurat package was applied to integrate the complete data set. The genes enriched in each cluster were identified using FindAllMarkers function in Seurat. It applies a Wilcoxon Rank Sum test and then performs multiple test correction using the Bonferroni method. The multiple-test corrected P < 0.05 was used as cut-off for significance.

Fresh human skin tissue was kept in MACS Tissue Storage Solution (Miltenyi Biotec, cat. no. 130-100-008) at most overnight. It was then enzymatically and mechanically dissociated using the Whole Skin Dissociation kit for human material (Miltenyi Biotec, cat. no. 130-101-540) and the Gentle MACS Dissociator device (Miltenyi Biotec), following the manufacturer’s instructions. The resulting cell suspensions were filtered through 70 µm cell strainers (Falcon, cat. no. 10788201) and the Dead Cell Removal Kit (Miltenyi Biotec, cat. no. 130-090-101) was then used to remove apoptotic and dead cells. Subsequently, about 20,000 single cells per sample were used to prepare sequencing libraries with the Chromium Single Cell Reagent kit (v3.1 chemistry) from 10X Genomics (cat. no. 1000128), following the manufacturer’s instructions. Library quality was checked with the Qubit dsDNA HS Assay kit (Life Technologies, cat. no. Q32851), and cDNA integrity was assessed with D1000 ScreenTapes (Agilent Technologies, cat. no. 5067-5582). Finally, pairwise sequencing (26 + 96 bp) was performed with a NovaSeq 6000 device (Illumina).

E14 MKO/tdT and Het/tdT embryos were collected, decapitated and eviscerated. To generate single cells, truncated embryos were incubated in 0.2% (wt/vol) collagenase‐type XI (Sigma‐Aldrich) and 2.4 U mL⁻¹ dispase II (Invitrogen) in DMEM at 37 ℃ 30 min, followed by filtering through a 40‐µm cell strainer. Cells were then centrifuged, resuspended in cold 5% FBS in PBS, and immediately sorted with a Beckman moflo Astrios EQ FACS sorter. Endogenous tdT signal was detected through PE channel and gates were set up according to the positive and negative controls. Sorted tdT+ cells were resuspended in 20% FBS/PBS buffer and loaded onto a droplet‐based library prep platform Chromium (10x Genomics) with a Chromium Single Cell Reagent Kit (version 3) according to the manuscript's instructions. Sequencing was performed on an Illumina nova 6000 and 150‐base pair paired‐end reads. The sequencing and bioinformatics analysis were performed by OE Biotech Co., Ltd. (Shanghai, China). All statistical analyses, unless otherwise specified, were conducted using R.^[32]