Illumina NovaSeq 6000 sequencing; Single-Cell Transcriptomics Sheds Light on the Identity and Metabolism of Developing Arabidopsis Leaf Cells
Single-Cell Transcriptomics Sheds Light on the Identity and Metabolism of Developing Arabidopsis Leaf Cells
Protoplasting protocol was adapted from (Ryu et al., 2019). Briefly, the third leaf of 40 plants per sample was harvested, chopped and incubated in 2 ml enzyme solution (0.4 M mannitol, 10 mM CaCl2, 20 mM MES, 20 mM KCl, 2% (wt/vol) cellulase and 0.5% (wt/vol) macero enzyme) constantly shaking for 30 min at room temperature. After a 7-min centrifugation at 100 g, the pellet was resuspended in 1 ml washing solution (0.4 M mannitol, 10 mM CaCl2, 20 mM MES and 20 mM KCl). Resuspended cells were filtered through a 50-μm cell strainer, which was washed with 0.5 ml of washing solution. Cells were sorted on the Melody FACS, where gates to sort out debris of dead cells were pre-defined via a test sample that was DAPI-stained (viable cell ratio 31%, prior to sorting). Subsequently, 100,000 cells per sample were sorted in less than 10 minutes. Arabidopsis Col-0 was grown under a long-day regime (16h-light and 8h-dark, intensity 110–120 μmol m−2s−1) at 21 °C on the WIWAM platform (http://www.wiwam.be). Five seedlings were grown per pot in order to obtain 40 seedlings per sample. The seeds were sown in 85±1 g of Saniflor compost (Van Isreal N.V., Geraardsbergen, Belgium) with an average absolute water content of 70%. Plants were watered daily until 8 days after stratification (DAS) with a well‐watered regime of soil relative water content (RWC) 69% (2.2 gwater/gsoil). At 9 DAS, half of the pots (randomized) were not watered until harvest (14 DAS) while the other half kept the well-watered regime. Single-cell suspensions were loaded on a GemCode Chromium Instrument (10X Genomics) to generate single-cell gel beads-in-emulsion (GEMs) using the Chromium Single Cell 3' Gel Bead and Library Kit, version 3 (10X Genomics, Cat. 1000075) and the Chromium i7 Multiplex Kit (10x Genomics, Cat. 120262) according to the manufacturer's instructions. In brief, reverse-transcription was performed in a 96-deep-well reaction module at 53°C for 45 min, 85°C for 5 min and ending at 4°C. Next, GEMs were broken and cDNA was cleaned up with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific, Cat. 37002D) and SPRIselect Reagent Kit (Beckman Coulter, No. B23318). Full-length, barcoded cDNA was amplified at 98°C for 3 min, 12 cycles at 98°C for 15 s, 67°C for 20 s and 72°C for 1 min, 1 cycle at 72°C for 1 min and ending at 4°C.  Illumina-ready sequencing libraries were prepared after clean-up with the SPRIselect Reagent Kit and enzymatic fragmentation via end-repair, A-tailing, adapter ligation, post-ligation SPRIselect clean-up and sample index PCR using the Chromium Single Cell 3' Library Kit (10X Genomics, Cat 1000078). The cDNA content of pre-fragmentation and post-sample index PCR samples was analyzed using the 2100 BioAnalyzer (Agilent).