Gel bead multiplex kit

scRNA-seq was performed on PBMCs from 26 COVID-19 patients. 14 WARD patients and 12 ICU patients (all upon admission except for one at discharge) were included. Single-cell suspensions were converted to barcoded scRNA-seq libraries by using the Chromium Single Cell 5’ library and Gel Bead & Multiplex Kit from 10x Genomics. Captured cells were lysed and the released RNA was barcoded. Libraries were sequenced on an Illumina NovaSeq 6000 sequencer according to a paired-end reading strategy. Raw gene expression matrices were generated per sample by the Cell Ranger pipeline using the human reference version GRCh38. Additionally, we processed scRNA-seq data from Wilk et al. (17 (link)), including 6 HCs, 4 severe, and 4 moderate COVID patients. All datasets were analysed by the Seurat package and merged using Harmony. The R package Slingshot was used to explore pseudotime trajectories (34 (link)).

Single-cell RNA sequencing was performed on 13 ‘mild-moderate’ and 10 ‘critical’ disease PBMC samples as well as 11 fresh BAL samples, sequencing 60675, 22849 and 26605 cells, respectively (Suppl. Figs. 4a and 8a). Single-cell suspensions were converted to barcoded scRNA-seq libraries by using the Chromium Single Cell 5′ library and Gel Bead & Multiplex Kit from 10x Genomics. Libraries were sequenced on an Illumina NovaSeq6000, and mapped to the human genome GRCh38 using CellRanger (10x Genomics). Raw gene-expression matrices generated per sample were merged and analysed with the Seurat package (v3.1.4).

The single-cell encapsulation and library preparation was done at the Centre for PanorOmic Sciences of the University of Hong Kong. The single-cell suspension was converted to uniquely barcoded RNA, TCR and BCR libraries by using the Chromium Single Cell 5′ Library, Gel Bead & Multiplex Kit, Chromium Single Cell V(D)J Enrichment Kit (Human T Cell), Chromium Single Cell V(D)J Enrichment Kit (Human B Cell), 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 CellRanger (10× Genomics). Gene positions were annotated as per Ensembl build 85 and filtered for biotype (only protein-coding, long intergenic noncoding RNA, antisense, immunoglobulin, T-cell receptor, and B-cell receptor).

Libraries for scRNA-seq were generated using the Chromium Single Cell 3′ library and Gel Bead & Multiplex Kit from 10x Genomics. 10×Genomics Chromium barcoding system was used to construct a 10× barcoded cDNA library following the manufacturer’s instructions. All libraries were sequenced on Illumina HiSeq 4000 until sufficient saturation was reached.

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.

The 10X Genomics Chromium platform (10X genomics, Pleasanton, California, USA) was used to capture and barcode the cells to generate single-cell Gel Beads-in-Emulsion (GEMs) by following the manufacturer’s protocol. Chromium Single Cell 3' V2 Chemistry Library Kit, Gel Bead & Multiplex Kit, and Chip Kit from 10X Genomics (Pleasanton, California, USA) were used as a standard procedure of the 3-terminal single-cell transcription sequencing scheme. After cDNA amplification and Library construction, Qubit and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) were used to measure the concentration. After the database was built, Illumina HiSeq X10 (Illumina, San Diego, CA, USA) was used for sequencing.