Novaseq 6000 sequencer

Single‐cell suspensions were obtained as previously described.⁴³ Briefly, fresh tumour samples were cut into pieces and digested with a MACS tumour dissociation kit (Miltenyi Biotec). Single‐cell suspensions were collected, and a Rigel S3 fluorescence cell analyser (Countstar) was used to assess cell viability and concentration. The cell suspension was loaded onto the Chromium single‐cell controller (10x Genomics). The scRNA‐seq libraries were constructed using the Single Cell 3′ Library and Gel Bead Kit v3.1 and sequenced using the Illumina NovaSeq 6000 sequencer (performed by CapitalBio Technology). scRNA‐seq data were aligned and quantified using the CellRanger toolkit v.3.1. Major cell types in the tumour tissue were clustered using Seurat (v.3.2.3). Differentially expressed genes were identified using FindMarkers. Enrichment analysis was performed using KOBAS software with Benjamini‒Hochberg multiple testing adjustment using the top 20 marker genes of the clusters. The outcomes were graphically represented utilizing the R software package.

The cultured WJ‐MSCs (huc_1 P0, huc_1 P3, huc_2 P0, huc_2 P3, huc_3 P0, and huc_3 P3) were digested to single‐cell suspension and adjusted to ≈1.0 × 10⁶ cells/mL. In accordance with the 10× Genomics recommended protocol, scRNA‐seq was performed with the Chromium 3′ V3 Chemistry (10× Genomics). An Illumina Novaseq6000 sequencer was used to sequence the single‐cell RNA‐seq libraries (performed by CapitalBio Technology, Beijing). Raw sequencing data was processed using the cellranger‐3.1.0. Feature‐barcode matrix generation and clustering were performed with Cell Ranger count module through alignment, filtering, and barcode counting.

Libraries for scRNA-seq were prepared using the Chromium Single Cell Platform with a Single-Cell 5′ Library and Gel Bead Kit (10X Genomics, PE-1000006). Thawed PBMC samples were evaluated for viability prior to scRNA-seq analysis, and all were ≥95% viable. Cells were resuspended in a volume equivalent to 10,000 target cells for each sample, and were individually loaded onto a Chromium single-cell controller (10X Genomics) to generate single-cell gel beads-in-emulsion (GEMs). Captured cells were then lysed and the released RNA was barcoded through reverse transcription in individual GEMs. Complementary DNAs (cDNA) were generated and split to generate additional libraries γδ scTCR-seq amplicons. To this end, gene-specific primers^{25 (link)} were used within the 5′ regions of the TRGC and TRDC segments for the enrichment of TCR transcripts.
Complementary DNAs were amplified, and the quality was assessed using an Agilent 4200 Tapestation.
The scRNA-seq libraries were sequenced using an Illumina Novaseq 6000 sequencer with a paired-end 150-bp (PE150) reading strategy (performed by CapitalBio Technology) and the scTCR-seq libraries were sequenced on Illumina NextSeq 550 platform.