scRNA-seq WT v2
The equal number of ovules at five stages (-2, -1, 0, 1, and 2 DPA) were harvested from different cotton bolls followed by partial tissue enzymatic digestion (PTED). The ovules were digested with 1.5% cellulase R-10 (L0012, Yakult, Japan), 1.0% hemicellulose (H2125, Sigma-Aldrich, US.), 0.3% Macerozyme R-10 (L0021, Yakult, Japan) in 10 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 0.1% BSA, 2 mM MES pH 5.7, and 600 mM mannitol for 1.5 hours rotation (~50 rpm) at 28C. The ovules were then filtered with a 40 m Falcon nylon mesh (Fisher scientific, US.) to remove undigested tissues and other large impurities. The filtrate was centrifuged for 10 mins at 100 g. The protoplast pellet was retained and washed 3 times with 8% mannitol, and finally resuspended in 500 L 8% mannitol buffer. The number of protoplasts was counted using a hemocytometer. After trypan blue staining, only the protoplast sample with more than 85% viability was used.  The scRNA-seq libraries were prepared using the Chromium Single Cell Gel Bead and 3 Reagent Kit v2 and v3 (10(Genomics, Pleasanton, CA). About 15,000-20,000 cells were loaded in the 10(Genomics Chromium single-cell microfluidics (10(Genomics, Pleasanton, CA) to generate single-cell gel beads in emulsion (GEMs). After size and quality check with Agilent 2100 Bioanalyzer, the libraries were sequenced on BGISEQ-500 (BGI, China) in PE100 mode.