title: GSM9313107: 5 days mock-inoculated soybean roots, rep1, scatac; Glycine max; ATAC-seq
library_name: GSM9313107
library_construction_protocol: Briefly, root tissues were finely chopped   with single edge razor blades on ice for approximately 2 minutes in 600 μL of pre-chilled Nuclei Isolation Buffer (NIB; 10 mM MES-KOH, pH 5.4; 10 mM NaCl; 250 mM sucrose; 0.1 mM spermine; 0.5 mM spermidine; 1 mM DTT; 1% BSA; and 0.5% Triton X-100). The homogenate was passed through a 40-μm cell strainer and centrifuged at 500 × g for 5 minutes at 4 °C. The supernatant was discarded, and the pellet was gently resuspended in 500 μL of NIB wash buffer (10 mM MES-KOH, pH 5.4; 10 mM NaCl; 250 mM sucrose; 0.1 mM spermine; 0.5 mM spermidine; 1 mM DTT; and 1% BSA). The suspension was filtered through a 40-μm cell strainer and carefully layered onto 1 mL of 35% Percoll (prepared by mixing 35% Percoll with 65% NIB wash buffer) in a 1.5-mL centrifuge tube. Nuclei were enriched by centrifugation at 500 × g for 10 minutes at 4 °C. Following centrifugation, the supernatant was removed, and the nuclei pellet was resuspended in 10 μL of diluted nuclei buffer (DNB; 10x Genomics, Cat# 2000207). For quality control, 5 μL of the nuclei suspension were diluted tenfold, stained with DAPI (Sigma, Cat# D9542), and examined under a fluorescence microscope using a hemocytometer to assess integrity and concentration. The nuclei concentration was then adjusted to 3,200 nuclei/μL using DNB buffer, and 5 μL (approximately 16,000 nuclei) were used as input for single-cell ATAC-seq library construction. Single-cell ATAC-seq libraries were generated using the Chromium Next GEM Single Cell ATAC v1.1 kit (10x Genomics, Cat# 1000175) according to the manufacturer's protocol (Chromium Next GEM Single Cell ATAC Reagent Kits v1.1 User Guide, CG000209 Rev E). The resulting libraries were sequenced on an Illumina NovaSeq 6000 platform using dual-index mode with 8 cycles for the i7 index and 16 cycles for the i5 index.