bulk RNA-seq of germinating Arabidopsis thaliana seeds with and without protoplasting
bulk RNA-seq of germinating Arabidopsis thaliana seeds with and without protoplasting
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. For bulk RNA-seq, embryos either collected without protoplasting (np) or with protoplasting (p).  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 1ml 60% Percoll cushion. Sample was centrifuged at 800g for 5min and fraction of embryos (at the bottom of the tube) was collected and re-suspended in 0.6ml 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, 20mM MES, 20mM KCl in milli-Q water and adjust the pH to 5.7 with 1M Tris/HCL pH7.5, 0.1% w/v BSA, 10mM CaCl2, and 5mM β-mercaptoethanol). After 3 hour of digestion, protoplasts were filtered through a 70μm cell strainer, then 40μm cell strainer to remove debris and centrifuged at 500g for 5min. Supernatant was removed and pellet was washed with 2ml protoplasting buffer without enzymes and mercaptoethanol and centrifuged at 500g for 5min. Pellet was resuspended in 50μl 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 Libraries were prepared using TruSeq Stranded Total RNA with Ribo-Zero Plant kit.