A protocol for time-resolved transcriptomics through metabolic labeling and combinatorial indexing

Abstract

The snapshot nature of single-cell transcriptomics presents a challenge for studying the dynamics of gene expression. Metabolic labeling, where nascent RNA is labeled with 4-thiouridine (4sU), captures temporal information at the single-cell level, providing greater insight into expression dynamics. Here, we present an optimized, automation-friendly protocol for the metabolic labeling of RNA alongside single-cell RNA sequencing through combinatorial indexing. We describe steps for 4sU labeling, cell fixation and chemical treatment, and automated two-level combinatorial indexing. For complete details on the use and execution of this protocol, please refer to Maizels et al.¹.

Keywords: Cell culture; Developmental biology; Gene Expression; Molecular Biology; Molecular/Chemical Probes; RNAseq; Single Cell.

Graphical abstract

Figure 1

Overview of combinatorial indexing Cells are distributed into multi-well plates where they receive well-specific barcodes. Each individual barcode is given to multiple cells, but through the sequential incorporation of random barcodes, each individual cell should receive a unique combination of barcodes, providing single-cell resolution. For example, with two rounds of 384 barcodes, there are 147,456 possible barcode combinations, allowing over ten thousand cells to be sequenced per experiment with a ∼6% doublet rate. Reverse transcription is performed in the first plate with barcoded primers, then library preparation is completed in a second plate, with barcoded primers used for PCR amplification.

Figure 2

Examples of BioAnalyzer traces of final library products (A) A suitable BioAnalyzer trace. (B) A BioAnalyzer trace showing primer-dimer contamination (red arrow). (C) A BioAnalyzer trace showing high molecular weight contamination, from overamplification or genomic DNA contamination.

Figure 3

UMIs per barcode/cell, ordering barcodes from highest to lowest UMIs, shown on a log-log scale A sharp elbow shows the transition from real cellular content to ambient RNA, occurring just before the theoretical maximum number of cells, fifty (red dotted line).