Chromium single cell controller

Each 10,000 nuclei were used for the snRNA or snATAC library construction.
For snRNA-seq, the single cell 3′ GEM, Library & Gel Bead Kit V3.1 (10× Genomics, 1000075) and Chromium Single Cell B Chip Kit (10× Genomics, 1000074) were used. To generate single-nuclei gel beads in emulsion, the nuclei suspension was loaded onto the Chromium single cell controller (10× Genomics). Then suspended the single nuclei in PBS (containing 0.04% BSA). Captured cells were lysed to release their RNA and barcoded through reverse transcription in individual GEMs. The reverse transcription was performed on a S1000TM Touch Thermal Cycler (Bio-Rad) at 53 °C for 45 min, followed by 85 °C for 5 min. The cDNA was kept at 4 °C and then amplified for sequencing.
For snATAC-seq, incubating the nuclei with Tn5 transposase. Then the nuclei suspension was loaded into the Chromium microfluidic chip E with 10x Genomics reagents and barcoded with a 10x Genomics Chromium Controller (10x Genomics, Pleasanton, CA). DNA fragments were subsequently amplified, and the sequencing libraries were constructed with reagents from a Chromium Single Cell ATAC reagent kit (10x Genomics; PN-1000110, PN-1000156, PN-1000084) according to the manufacturer’s instructions. After preliminary quantification and quality inspection, libraries were then pooled and loaded on an Illumina NovaSeq with 2 × 50 paired-end kits.

ScRNA-seq libraries were prepared according to previous protocols. In brief, the recovered PBMC were counted in 0.4% trypan blued, centrifuged and re-suspended at the concentration of 2 × 10⁶/ml. The cell suspension was loaded onto a Chromium single cell controller (10× Genomics) to generate single-cell gel beads in the emulsion (GEMs) according to the manufacturer’s protocol. Reverse transcription takes place inside each GEM, after which cDNAs are pooled together for amplification and library construction. The resulting library products consist of Illumina adapters and sample indices, allowing pooling and sequencing of multiple libraries on the next-generation short read sequencer.

scRNA-seq library generation was described in our previous published study^{37 (link)}. Two biological repeats for each samples (including iNKT cells from spleen, liver, and lymph node) of scRNA-seq libraries were generated using the 10X Genomics Chromium Single Cell 3′ Reagent Kit (v2 Chemistry) and Chromium Single Cell Controller as previously described^{38 (link)}.

IMRCs and UCMSCs were harvested and suspended in PBS. Then, cell suspensions were loaded onto the Chromium Single Cell Controller (10 × Genomics) to generate single Gel Beads-in-Emulsion (GEMs) by using Single Cell 30 Library and Gel Bead Kit V2 (10 × Genomics, 120237). Cells were lysed and the released RNA was barcoded through reverse transcription in individual GEMs. Following reverse transcription, cDNAs with both barcodes were amplified, and a library was constructed using the Single Cell 30 Reagent Kit (v2 chemistry) for each sample following the manufacture’s introduction (performed by CapitalBio Technology, Beijing). Sequencing was performed on an Illumina NovaSeq 6000 System in the 2 × 150 bp paired-end mode. Raw files were processed with Cell Ranger using default mapping arguments. Gene expression analysis and cell type identification were performed by Seurat V3.1. Briefly, after normalizing and quality control, the tSNE was used for non-linear dimensional reduction. The figures were produced by DimPlot and VlnPlot functions in Seurat.

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.

Previously method 31 (link) were performed by CapitalBio Technology, Beijing. Briefly, the cell suspension was loaded onto the Chromium single cell controller (10× Genomics) to generate single-cell gel beads. Captured cells were lysed, and RNA were barcoded through reverse transcription and finally the cDNA was generated. Single-cell RNA-seq libraries were prepared using Single Cell 3' Library and Gel Bead Kit V3, and sequenced using an Illumina Novaseq6000 sequencer with a sequencing depth of at least 100,000 reads per cell with pair-end 150 bp (PE150) reading strategy.