Single cell RNA-seq of BHK-21 cells carrying alphaviral RNA replication system

Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. Toinvestigatetheexpressionheterogeneityofself-replicatingRNAsinrepRNA-v4cells,weperformedsingle-cellRNA-seqanalysisusingthe10xGenomicssequencingmethod.Ouranalysisofthesingle-cellRNA-seqprofilingdatarevealedarelativelyuniformexpressionpatternofself-replicatingRNAswithinrepRNA-v4cells. In this study, 10 μg of repRNA-v4 RNA was electroporated into 4 million BHK-21 cells expressing nsP4 in trans. The cells were cultured for ~3 weeks in the presence of 10 μg/ml of puromycin. ~20000 cells were used for the preparation of 10x Genomics 5' gene expression libraries. Subsequently, RNA-seq was performed on an Illumina's high-throughput sequencing platform NovaSeq 6000. Cleaned reads were demultiplexed, barcode processed, gene counted using the Cell Ranger software v4.0.0. against the Mesocricetus auratus genome (GCF_017639785.1_BCM_Maur_2.0_genomic.fna) and the genome of alphaviral RNA replication system. The genome annotation file (GCF_017639785.1_BCM_Maur_2.0_genomic.gff) was downloaded from NCBI and modified to include annotation about the genome of the alphaviral RNA replication system. Subsequent analyses were performed using the Seurat package with default or recommended parameters.