The utilization of single cell RNA sequencing (RNA-seq) has made remarkable progress in various fields. Researchers benefit from its provision of ultrahigh resolution when looking into the transcriptome regulation. In many cases, the sample requires fixation due to long-term transportation or in specially designed experiments. Methanol fixation has been widely used in single cell RNA-seq for its simple operation and minimal influence on cellular structure. Due to the vulnerability of mRNA, the fixation process may cause changes on the RNA sequencing profile, which will bias the downstream analysis. In this study, we adopted a fixation protocol from previous study and performed single cell RNA-seq on both live and methanol fixed cells. By comparing the sequencing result thorough...[ Read more ]

The utilization of single cell RNA sequencing (RNA-seq) has made remarkable progress in various fields. Researchers benefit from its provision of ultrahigh resolution when looking into the transcriptome regulation. In many cases, the sample requires fixation due to long-term transportation or in specially designed experiments. Methanol fixation has been widely used in single cell RNA-seq for its simple operation and minimal influence on cellular structure. Due to the vulnerability of mRNA, the fixation process may cause changes on the RNA sequencing profile, which will bias the downstream analysis. In this study, we adopted a fixation protocol from previous study and performed single cell RNA-seq on both live and methanol fixed cells. By comparing the sequencing result thoroughly, we conclude that although subtle alterations can be introduced because of a specific mRNA subpopulation, methanol fixation can preserve the biological meaning of single cells to a large extent. By elucidating the pros and cons of methanol fixation, researchers will better understand and interpret their sequencing result.