scATAC-seq WT rep1
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.  For scATAC-seq, the fresh protoplasts were lysed with 10 mM Tris-HCL, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 0.1% NP-40, 0.5% Dodecyl Maltoside (DDM), and filtered with 40 m nylon mesh (CORNING, US.). The nuclei were then spun down with 500 g for 3 mins, resuspended, and purified using SH800S flow cytometer (Sony, Japan). For each sample, about 12,000 nuclei were used for Tn5 tagmentation and amplification following the previous protocol (Buenrostro et al., 2015). The MGISEQ-2000 (BGI, China) with PE50 mode was used for sequencing the libraries.