Chromium single cell chip kit

Early-stage orthotopic tumours derived from PARD3-overexpressing and wild-type (WT) Hepa1-6 cells were excised from C57BL/6J mice 10 days after tumour cell implantation. Tumour tissues were digested in 8 mg/10 ml collagenase IV in PBS at 37 °C for 30 min. The undigested pellets were discarded, and cells in the supernatant were collected by centrifugation at 400 × g for 5 min. The cell pellets were resuspended in PBS containing 0.5% BSA to obtain single-cell suspensions before single-cell encapsulation and library preparation, which was performed at the Centre for PanorOmic Sciences, The University of Hong Kong. Uniquely individually barcoded RNA was obtained by using the Chromium Single Cell 3′ Library, Gel Bead & Multiplex Kit, and Chromium Single Cell Chip Kit following the instructions provided by 10x Genomics. RNA-seq was performed on the NovaSeq 6000 platform. Chromium single-cell data were processed with Cell Ranger software (v 6.1.2, 10x Genomics) to align reads and generate feature-barcode matrices. The Seurat object was constructed based on the gene expression matrix and metadata using the Seurat R package (v 4.0). In the quality control (QC) process, single cells with read counts > 5000 or < 200 and > 20% mitochondrial read counts were excluded from subsequent downstream analyses.

Single cells were processed through 10X Chromium system (10X Genomics, USA) using the single-cell 5’ library and the gel bead kit (10X Genomics, PN-1000006), and the Chromium single cell chip kit (10X Genomics, PN-1000151) as per the manufacturer’s instructions. The cells were partitioned into barcoded gel bead‐in‐emulsions reverse transcription was performed on individual droplets. cDNA libraries were then sequenced using an Illumina Hi-Seq 2500/NOVA (Illumina).

The PBMCs co-cultured with C666-NC and C666-KO NPC cells after 48 hours were isolated from the culture medium by centrifuging at 300× g for 5 mins, and re-suspended in PBS before single-cell encapsulation. The single-cell encapsulation and library preparation was done at the Centre for PanorOmic Sciences, the University of Hong Kong. The single-cell suspension was converted to uniquely barcoded RNA, using the Chromium single cell 3′ library, gel Bead & multiplex kit, and Chromium single cell chip kit (10× Genomics), as per manufacturer’s instructions. The libraries were sequenced on a NovaSeq 6000, and mapped to the human genome (hg38) using Cell Ranger (v 6.1.2, 10x Genomics). Gene positions were annotated as per Ensembl build 85 and filtered for biotype. The sample aggregation and analysis were performed using the aforementioned methods.