title: GSM8939989: snRNA-seq from seed nuclei, day after pollination 3, replicate 1b; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Two biological replicates, (different plants hand pollinated and processed for snRNA-seq on different days) each containing seeds dissected from 10-15 siliques (500-800 seeds), were collected for each timepoint, producing six replicates total. Timed seeds were dissected into 150uL cold extraction buffer on ice (1x Partec nuclei extraction buffer, 4% BSA, 1mM DTT, 1:100 protease inhibitor cocktail for plants, and 1:30 Protector RNase inhibitor). Seeds were mechanically dissociated in 1.5mL tubes by grinding with an Axygen pestle for 10 turns. The nuclei suspension was filtered through a 30um cell strainer prewet with Partec nuclei staining buffer into a 5mL tube for fluorescence activated nuclei sorting (FANS). The strainer was rinsed with Partec nuclei staining buffer into the 5mL tube to bring the final suspension volume to 1mL. 1uL of 1mg/mL DAPI was added to increase nuclear signal. Nuclei were purified and concentrated by FANS on a BD FACS ARIA Cell Sorter using a 70um chip. We gated on 2N, 3N, 4N, 6N, 8N, and 16N peaks (Suppmentary Fig. 6). Nuclei were sorted into 30-50uL collection buffer (PBS-4% BSA) in a 1.5mL tube, and concentration was assessed using the ARIA nuclei count and by manual counting on a Neubauer Improved hemocytometer. Each biological replicate was processed individually using one Chromium Next GEM Single Cell 3' Kit (v3.1) reaction with a target nuclei recovery of 10k, except for one 3DAP biological replicate, which was split across two reactions. In total, 7 libraries were generated in this study. Chromium Next GEM Single Cell 3' Kit (v3.1), Illumina