Single-cell RNA Seq on DNBSEQ-T1+ Using Bio-Rad ddSEQ 3’ RNA-seq Kit

Introduction

Single-cell RNA sequencing (scRNA-seq) has transformed the study of complex tissues by resolving cellular heterogeneity, transient states, and lineage trajectories that are obscured by bulk approaches [1]. As single-cell studies scale from thousands to millions of cells, sequencing workflows must deliver sensitivity to low-abundance transcripts, multiplet and crosstalk control, flexible throughput, and cost-efficient scalability.

The Bio-Rad ddSEQ Single-Cell 3’ RNA-Seq Kit is a droplet-based workflow that uses barcoded beads with deconvolution oligos to maximize usable cell recovery. It supports 500–10,000 input cells or nuclei, enables same-day cDNA and deconvolution-oligo library generation with multiple safe stopping points, and includes open-source Omnition analysis for rapid QC and reporting.

The Complete Genomics DNBSEQ-T1+ sequencer, powered by DNBSEQ™ technology, provides flexible mid-throughput sequencing with high Q40 accuracy and output ranging from 50Gb to 1.2Tb in under 24 hours.

Methods

Sample and Library Preparation​

Multiple independent experiments were performed using A375, A549, and mixed-species HEK293/NIH 3T3 samples loaded onto the ddSEQ Single-Cell Isolator at various target outputs. Libraries were prepared with the ddSEQ 3’ scRNA-Seq Kit, generating cDNA and deconvolution oligo libraries, both containing P5 and P7 adapters for dual-indexed paired-end sequencing.​

Library Conversion and Sequencing​

ddSEQ 3’ scRNA-seq libraries were split for sequencing on the Complete Genomics DNBSEQ-T1+ and Illumina NextSeq 2000 platforms. For DNBSEQ-T1+ sequencing, libraries were converted to single-stranded circles using the DNBSEQ Universal Library Conversion Kit. cDNA libraries were converted individually, while DO libraries were pooled before conversion. Resulting cDNA and DO DNA nanoballs were pooled with a 35% standard-library spike-in to ensure base diversity and sequenced on the DNBSEQ-T1+ using the High-Throughput Sequencing Reagent Set, FCL PE150.​

Analysis​

FASTQ files from both platforms were analyzed with Omnition Analysis Software v1.1.0 using default parameters. DNBSEQ-T1+ FASTQ filenames were modified for Omnition compatibility. Sequencing data were downsampled to 40,000 or 50,000 reads per cell for comparison, and tertiary analyses were performed using Seurat v5.0.3​