An improved nuclei isolation protocol from leaf tissue for single-cell transcriptomics
We developed an alternative method for isolating nuclei to be utilized in scRNA-seq experiments from Zea mays leaves with reduced chloroplast contamination. By effectively removing chloroplasts during the FACS step of our protocol, using the autofluorescence from the chloroplasts, we achieved improved alignment of reads to the genome and transcriptome. Our enhanced protocol offers a valuable solution for applying snRNA-seq in tissues with a high content of chloroplasts. Overall design: After nuclei isolation following Conde et al (2021), we used a FACS strategy for clean-up and enrichment of the nuclei using a BD FACSAriaTM IIU/III upgraded cell sorter. We used a double-filter strategy to negatively select autofluorescent chloroplast and positively select DAPI-stained nuclei (Fig 1). The Peridinin-Chlorophyll-Protein (PerCP) filter, excited by the 488 nm blue laser and captured with a 670/30 nm bandpass filter, was used to remove the events that emitted in this range. After this, the DAPI-positive population was selected according to emission in the 450/50 nm bandpass filter and then based on size and granularity using the forward versus side scatter (FSC vs SSC) plot. Finally, the selected population corresponding to nuclei was sorted, aiming for 40,000 nuclei for library preparation using 10X Genomics sc-RNAseq kit.