Extended Data Fig. 1: Supporting data for the generation of the fixation and digestion at high temperatures protocol.
a, Representative image showing the released cells from different tissues and species. Varying plant tissues were digested with the fixation and digestion at high temperatures protocol. See quantification results in Fig. 1c. b, Images showing the cells prepared from fixed rice tiller nodes, wild rice rhizome nodes, and shoot apices of Selaginella martensii. The tissues were subjected to fixation and digestion at high temperatures using a combination of two enzymes (that is, reduced enzyme mix, C + M) or four enzymes (C + M + S + P). Red arrows indicate single cells. Please note that the number of released cells was greatly increased when tissues were digested by a combination of four enzymes. Scale bars, 50 μm. See quantification results in Fig. 1d. c, Quantification of released protoplasts or cells from maize root tips. We compared the optimized protoplasting protocol from Ortiz-Ramirez et al.53 and our fixation-based protocol. Maize root tips were digested following Ortiz-Ramírez et al. and released protoplasts were quantified via hemocytometer (grey bar, 1). Maize root tips were fixed then digested at 50 °C for 90 min using the Ortiz-Ramírez et al. (2018) protoplast enzyme mix (blue bar, 2) or reduced enzyme mix (blue bar, 3). The released cells were similarly quantified. Data are the mean ± s.e.m. from five independent biological replicates. Different letters denote statistically significant variation (two-sided Student’s t-test, p < 0.05, with no adjustment for multiple comparisons). The p-values are provided in Source Data. d, The addition of vanadyl ribonucleoside complexes (VRC) leads to production of black precipitates (red arrow) in the digestion solution. e, Assessment of RNase enzymatic activity in the digestion buffer. We evaluated the effectiveness of commercial RNase inhibitors (Roche, Cat. No. 3335399001; New England Biolabs, Cat No. M0314L), EDTA, triGMP, and tRNA on the RNase activity of the original digestion enzyme blends. “purif.” indicates that RNase was removed from the digestion solution via affinity chromatography. “Blank” utilizes the enzyme-dissolving RNase binding buffer (RBB) as a control. Data are the mean ± s.d. from three independent experiments. f, Evaluation of the coupling efficiency of GMP onto NH2-Sepharose. The uncoupled NH2-Sepharose exhibited an absorbance of 5.6 Abs at 248 nm, while the NH2-Sepharose successfully coupled with GMP (GMP-Sepharose) displayed an absorbance of 10.1 Abs at 248 nm. g, Assessment of GMP-Sepharose column regeneration efficiency. We compared the performance (that is, RNase enzymatic activity in purified digestion enzyme blends) of columns that underwent regeneration (second purification with regeneration) versus those that did not undergo regeneration (second purification without regeneration). Data are the mean ± s.d. from three independent experiments. h, j, Assessment of RNA integrity through electrophoresis analysis. Rice roots were digested at various temperatures (h) and times (j). RNAs were extracted and subjected to electrophoresis analysis. Please note that RNA integrity decreased with increased time and temperature. On the far left is the DNA ladder. i, k, Assessment of RNA integrity through RIN analysis. Rice roots were digested at various temperatures (i) and times (k). RNAs were extracted and subjected to RIN analysis. Please note that RNA integrity decreased with increased time and temperature. Data are the mean ± s.d. from three independent experiments. Different letters denote statistically significant variation (two-sided Student’s t-test, p < 0.05, with no adjustment for multiple comparisons). The p-values are provided in Source Data.
