GSM9428752: Sample2, Crataegus scabrifolia, leaf Protoplasts; Crataegus scabrifolia; RNA-Seq
Protoplasts were isolated from cleaned hawthorn leaf material via an enzymatic digestion method. Five milliliters of enzymatic digestion solution (20 mM KCl, 10 mM CaCl₂, 0.1% bovine serum albumin, 20 mM MES, 0.6 M mannitol, 1% cellulase R10, 0.5% snailase, 0.5% macerozyme R-10) was added to the cleaned plant material fragments. The mixture was incubated at 25°C on a shaker at 40 rpm for 4–5 h to release protoplasts from the leaves. The protoplasts were filtered through a 40 µm cell sieve, centrifuged at 200 × g for 3 minutes, and washed with 2 mL of washing solution (5 mM KCl, 125 mM CaCl₂, 154 mM NaCl, 0.1% bovine serum albumin, 20 mM MES). An aliquot was stained with trypan blue and analyzed on a LUNA-FL Cell Counter to determine protoplast count and viability. Hawthorn leaf nuclei were isolated following protocol. Briefly, 1 mL of ice-cold NIB buffer (20 mM HEPES, pH 8.0, 250 mM sucrose, 1 mM MgCl₂, 5 mM KCl, 0.25% Triton X-100, 40% glycerol, 0.5 mM spermine, 0.5 mM spermidine, 0.1% 2-ME) was added to the cleaned plant material fragments, and the tissue was gently chopped with a razor for 5 min until it could be pipetted ,with 1 mL wide-bore tip. The homogenate was filtered through a 40 µm cell sieve into a chilled 50 mL conical tube. The filtrate was collected and washed with the remaining buffer to recover the nuclei. The mixture was incubated on ice for 10 min, centrifuged at 50 × g for 5 min at 4°C to remove bulk impurities, and then centrifuged at 1,100 × g for 10 min at 4°C. After the supernatant was removed, the nuclei were resuspended in 500 µL of NIB buffer, and the quality and count of the nuclei were validated via the Countstar Rigel S6 system with AOPI staining. Single protoplasts were captured via the BD Rhapsody HT single-cell analysis system, and scRNA libraries were constructed via the WTA Amplification Kit (BD). For snRNA library construction, single nuclei were captured via a 10X Genomics GemCode single-cell instrument with the Chromium Next GEM Single Cell 3' Reagent Kit (v3.1). The snRNA-seq and scRNA-seq libraries were constructed according to the protocols of each platform. Briefly, single protoplasts or nuclei were encapsulated in droplets or wells via HT Xpress chips (BD Rhapsody) or Chromium Next GEM Chip (10X Genomics) to create independent environments. mRNA was captured via magnetic beads, followed by protoplast/nuclear lysis, reverse transcription, cDNA amplification, purification, and conversion into single-cell/nuclear RNA-seq libraries with barcode sequences. Indexed sequencing libraries were prepared following the Illumina user guide, with each library assigned a unique index.