CEL-Seq2 is a highly sensitive, cost-effective, and user-friendly single-cell RNA-Seq method that improves upon the original CEL-Seq. It offers threefold higher sensitivity, lower costs, and reduced hands-on time. CEL-Seq2 was implemented on the Fluidigm C1 system, enabling on-chip barcoding and detecting gene expression changes during the cell cycle in mouse fibroblasts. It was also compared with Smart-Seq, demonstrating superior sensitivity. The method uses early barcoding and 3' end tagging to enhance accuracy and reduce the need for gene length correction. Key improvements include shorter primers, optimized reverse transcriptase and polymerase, and a more efficient library preparation process. These changes increase sensitivity and reduce noise in gene expression data. CEL-Seq2 is compatible with various platforms and allows for single library construction, reducing preparation time and cost. It was tested on mouse fibroblasts and dendritic cells, showing improved detection of transcripts and genes compared to other methods. CEL-Seq2 also provides better sensitivity and reproducibility than Smart-Seq, particularly in detecting low-abundance transcripts. The method uses unique molecular identifiers (UMIs) to improve accuracy and is compatible with different sequencing platforms. The data and pipeline are publicly available, enabling further research and application in single-cell transcriptomics.CEL-Seq2 is a highly sensitive, cost-effective, and user-friendly single-cell RNA-Seq method that improves upon the original CEL-Seq. It offers threefold higher sensitivity, lower costs, and reduced hands-on time. CEL-Seq2 was implemented on the Fluidigm C1 system, enabling on-chip barcoding and detecting gene expression changes during the cell cycle in mouse fibroblasts. It was also compared with Smart-Seq, demonstrating superior sensitivity. The method uses early barcoding and 3' end tagging to enhance accuracy and reduce the need for gene length correction. Key improvements include shorter primers, optimized reverse transcriptase and polymerase, and a more efficient library preparation process. These changes increase sensitivity and reduce noise in gene expression data. CEL-Seq2 is compatible with various platforms and allows for single library construction, reducing preparation time and cost. It was tested on mouse fibroblasts and dendritic cells, showing improved detection of transcripts and genes compared to other methods. CEL-Seq2 also provides better sensitivity and reproducibility than Smart-Seq, particularly in detecting low-abundance transcripts. The method uses unique molecular identifiers (UMIs) to improve accuracy and is compatible with different sequencing platforms. The data and pipeline are publicly available, enabling further research and application in single-cell transcriptomics.