scNT-seq2: single-cell metabolically labelled new RNA tagging sequencing for time-resolved analysis of gene expression in single cells v1

Single-cell metabolically labeled new RNA tagging sequencing (scNT-Seq) is a droplet microfluidics-based, time-resolved RNA-seq method for joint profiling of newly synthesized (“new”) and pre-existing (“old”) RNAs from the same cell by marking new transcripts with T-to-C substitutions via chemical conversion of 4-thiouridine (4sU) in new RNAs to cytidine analogs (Qiu et al., Nature Methods 17.10 (2020): 991-1001. PMID: 32868927). Here, we described the updated scNT-Seq2 method in which an optimized second-strand synthesis reaction is employed to increase the mapping rate and library complexity. This step-by-step protocol describes metabolic labeling, sample preparation, and scNT-Seq2 library preparation for Illumina sequencing.