NextSeq 500 sequencing; Single-cell RNA-seq of germinating Arabidopsis thaliana seeds
Single-cell RNA-seq of germinating Arabidopsis thaliana seeds
Col-0 seeds were sown and collected at the 12 h, 24 h and 48 h time-points, in three independent replicates (batches) of the experiment.  Col-0 seeds were sown on MS media plates (containing 3 % sucrose). Seeds were sterilised and stratified for 48 h of cold (4°C) dark stratification before being transferred to continuous light (at 22°C), then collected after 12 h, 24 h and 48 h in the light. Dissociation of Arabidopsis thaliana embryos into single cells: seeds were sandwiched between two glass slides and embryos were released mechanically from seed coats by pressing two slides. Ruptured seed coats and embryos were collected into microcentrifuge tubes and separated from each other using Percoll gradient. In brief, sample was resuspended in MC buffer (10mM potassium phospate pH7.0, 50mM NaCl, 0.1M sucrose) and loaded onto a 1 ml 60% Percoll cushion. Sample was centrifuged at 800 g for 5 min and fraction of embryos (at the bottom of the tube) was collected and re-suspended in 0.6 ml MC buffer. A second Percoll gradient was repeated to obtain pellet of embryos without any seed coats. Enriched embryos samples were cut with razor blades and digested in protoplasting buffer (2 % w/v Cellulase, 3 % w/v Macerozyme, 0.4M D-mannitol, 20~mM MES, 20~mM KCl in milli-Q water and adjust the pH to 5.7 with 1~M Tris/HCL pH7.5, 0.1 % w/v BSA, 10~mM CaCl2, and 5~mM β-mercaptoethanol). After 3 hour of digestion, protoplasts were filtered through a 70 μm cell strainer, then 40 μm cell strainer to remove debris and centrifuged at 500 g for 5 min. Supernatant was removed and pellet was washed with 2 ml protoplasting buffer without enzymes and mercaptoethanol and centrifuged at 500 g for 5 min. Pellet was resuspended in 50 μl protoplasting buffer without enzymes and mercaptoethanol. Protoplasts were counted using hemocytometer and adjusted to final concentration of 800-1200 protoplasts per μl. QIAgen Plant RNEasy kit 10x Genomics 3' end droplet-based single-cell RNA-seq (kit v2)