Currently available methods for transcriptome sequencing such as single-cell RNA-seq (scRNA-seq) are complex to perform. Here, the authors developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), which involves four rounds of combinatorial barcoding of cellular RNA. The technique involves a series of pipetting steps and requires no specialist equipment. Analysis of transcriptomes from developing mouse CNS neurons revealed that gene expression patterns corresponded to cellular function, developmental stage and regional specificity. This method allows scalable profiling of single neurons that allows analysis of complex tissues containing many cell types, thus improving the usefulness of this technique.