Chromium single cell controller

Single‐cell RNA‐seq was performed using the 10× Genomics Chromium Single Cell Controller with the Chromium Single Cell 3′ V2 Kit (Chromium Single Cell 3ʹ Library and Gel Bead Kit v2, 16 rxns PN‐120237, Chromium Single Cell A Chip Kit, 48 rxns PN‐120236, Chromium i7 Multiplex Kit and 96 rxns PN‐120262, all from 10× Genomics, USA) following the manufacturer's instructions with barcoded gel beads at a target capture rate of 10 000 individual cells per sample. After quality control, libraries were sequenced on the Illumina HiSeq X‐ten platform in 2 × 150 bp paired‐end mode.

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 Chromium Single Cell 3′ Library and Gel Bead Kit V2 (10x Genomics, 120237) and Single Cell A Chip Kit (10x Genomics, 120236) were used for cell capture. The cell suspension (300–600 living cells per microlitre, as determined by Count Star) was loaded onto the Chromium Single Cell Controller (10x Genomics) to generate single-cell gel beads in emulsion (GEMs) according to the manufacturer’s protocol. In short, single cells were suspended in PBS containing 0.04% BSA. Approximately 3173 cells were added to each channel, and the target cell recovery was estimated to be ~16,544 cells in total. Captured cells were lysed, and the released RNA was barcoded through reverse transcription in individual GEMs. 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 and a hold at 4 °C. cDNA was generated and then amplified, and quality was assessed using an Agilent 4200 (performed by CapitalBio Technology, Beijing).

Cells at different induction time points of BiPNT were collected and resuspended in DPBS with 0.04% BSA. Then, cells suspensions (500–1000 cells per microliter) were loaded on a Chromium Single Cell Controller (10x Genomics) to obtain single-cell gel beads in emulsion (GEMs) by using Single Cell 3' Library and Gel Bead Kit V2 (10x genomics, 120237). Captured cDNAs were lysed and the released RNA were barcoded through reverse transcription in singular GEMs. Barcoded cDNAs were pooled and cleaned by DynaBeads® MyOne Silane Beads (Invitrogen, 37002D). Single-cell RNA-seq libraries were prepared by Single Cell 3' Library Gel Bead Kit V2 (10x Genomics, 120237) following the manufacturer’s instruction. Sequencing was operated on an Illumina HiSeq X Ten with pair end 150 bp (PE150).