Chromium single cell 3 reagent kit

Single-cell suspensions were made using a gentle MACS Octo (Miltenyi Biotec). Single-cell suspensions were loaded onto the Chromium Controller (10× Genomics) for droplet formation. scRNA-seq libraries were prepared using the Chromium Single Cell 3′ Reagent Kit (10× Genomics). The cell suspension (300–600 living cells/mL) was loaded onto the Chromium single-cell controller (10× Genomics) by using the single-cell 3’ Library and Gel Bead Kit V3 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074). Details are shown in the Supplementary File. scRNA-seq libraries were constructed by the Single Cell 3’ Library and Gel Bead Kit V3. The libraries were sequenced by an Illumina Novaseq6000 sequencer, the sequencing depth of each cell was at least 100,000 reads, and the paired-end 150 bp (PE150) reading strategy (CapitalBio Technology, Beijing) was adopted. The Cell Ranger software was obtained from 10× Genomics website. Alignment, filtering, barcode counting, and UMI counting were performed with cell ranger count module. Dimensionality reduction and the first ten principal components were used to generate clusters by K-means algorithm and graph-based algorithm, respectively. Details of other analyses are shown in the Supplementary Files.

Immediately postsorting, CD45⁻ DAPI⁻ EpCAM⁺total TECs were run on the 10× Chromium and then single‐cell RNA‐seq libraries were generated using the Chromium Single Cell 3′ Reagent Kit (10× Genomics) by the core facility at the Embryology Department of Carnegie Institute for Science. Briefly, TEC single‐cell suspension (~1,000 cells per 1 µl PBS) was mixed thoroughly with Single Cell 3′ gel beads and partitioning oil into a Single Cell 3′ Chip (10× genomics) following the recommended protocol for the Chromium Single Cell 3′ Reagent Kit (v2 Chemistry). RNA transcripts of single cells were uniquely barcoded and reverse‐transcribed within the individual droplets. cDNA molecules were then pre‐amplified and pooled together followed by the final library construction. Libraries were sequenced by paired‐end 150‐bp reads on Illumina NextSeq 500. Postprocessing and quality control were performed by the same genomics core facility using the 10× Cell Ranger package (V2.1.1, 10× Genomics) as described by Zheng et al. (2017). Reads were aligned to mm10 reference assembly (v1.2.0, 10× Genomics). After cell demultiplexing and read counting, all three samples were combined using the cellranger aggr pipeline (Zheng et al., 2017). Cell visualization was done using the Loupe Cell Browser (10× Genomics).