title: Illumina NovaSeq 6000 sequencing; Single-Cell Transcriptomics Sheds Light on the Identity and Metabolism of Developing Arabidopsis Leaf Cells
design: Single-Cell Transcriptomics Sheds Light on the Identity and Metabolism of Developing Arabidopsis Leaf Cells
library_construction_protocol: 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).