Genome-wide DNA replication profiling and full-length total RNA sequencing from the same single cell [hTERT-RPE1 Whole-S]

Single-cell sequencing has advanced our understanding of cell-type diversity and heterogeneity. However, existing single-cell multi-omics methods lack DNA data resolution and full-length total RNA detection. Here, we introduce single-cell (sc)Repli-RamDA-seq (scRR-seq), a novel multi-omics method that enables high-resolution DNA replication profiling and full-length total RNA sequencing from the same single cell, made possible by the clean separation of DNA and RNA. scRR-seq generates DNA replication and RNA sequencing data comparable to individually obtained scRepli-seq and scRamDA-seq data, respectively. scRR-seq also allows one to tell the cell-cycle stage of a given S-phase cell and detects copy-number variation (CNV) in non-S-phase cells. scRR-seq outperforms other scRNA-seq methods in the number of transcripts identified and full-length total RNA detection. Furthermore, scRR-seq allowed haplotype-specific analysis and the identification of novel S-phase markers. Taken together, scRR-seq is a robust single-cell multi-omics method with promising potential for comprehensive genome and transcriptome analysis. Overall design: We performed simultaneous DNA-seq and RNA-seq on the same cells using the scRR-seq protocols (scRR1 and scRR3). The following cell types were used for the experiments: (1) hTERT-RPE1 cells collected from mid-S phase, (2) mouse 8-cell embryos, (3) CBMS1 mouse embryonic stem cells collected from the entire S phase, (4) hTERT-RPE1 cells collected from the entire S phase, (5) HAP1 cells collected from mid-S phase, and (6) IMR-90 cells treated with or without 0.4 µM aphidicoline, collected from the G1 phase. Single cells were collected into 0.2 ml PCR tubes by cell sorter or manually picked and lysed in RamDA-seq lysis buffer (TOYOBO, #RMD-101) containing 2 mg/ml Dynabeads™ MyOne™ Carboxylic Acid (Invitrogen, #65011). Genomic DNA was trapped on the beads, while RNA was released into the lysis buffer. The isolated DNA on magnetic beads was subjected to whole-genome amplification (WGA) and Next-Generation Sequencing (NGS) library preparation, as described in scRepli-seq (Takahashi et al., Nat Genet 2019). The DNA-seq (scRepli-seq) data was used to assess genome-wide DNA copy numbers, karyotype G1-phase cells, and analyze the replication state of S-phase cells. In parallel, the RNA isolated in the supernatant was subjected to scRamDA-seq (Hayashi et al., Nat Commun 2018) using the GenNext®RamDA-seq™ Single Cell Kit (TOYOBO, #RMD-101) according to the manufacturer's instructions with modifications.