title: GSM6599889: Nip-scATAC-Normal-Rep2; Oryza sativa; ATAC-seq
library_construction_protocol: Primary root tips with 5 mm in length from the 3-day-old rice seedlings were harvested and fixed with cold paraformaldehyde solution (2%) for 30 min. Then the root tips were transferred to Dounce grinder (kimble), which was filled with cold lysis buffer (10 mM Tris-HCl, 10 mM NaCl, 1 mM MgCl2, 1% BSA) and 0.2% Tween 20, 0.2% NP-40, 0.0075% Digitonin. 0.5% 2-ME (v/v) were added and then the roots were grinded completely. The homogenate was filtered with a 20 μm mesh (Sysmex) following by centrifuge at 480 rcf for 5 min at 4℃. After removing supernatant, 1% BSA buffer (10 mM Tris-HCl (pH7.4), 10 mM NaCl, 3 mM MgCl2, 1% BSA (v/v)) was added to resuspend pellet. Then, the mixture was placed on 80% percoll and following by centrifuge at 300 rcf for 25 min at 4℃. After removing 150 μl supernatant, nuclei were transferred to a new eppendorf tube and washed twice with 1% BSA buffer. The nuclei were resuspended in chilled Diluted Nuclei Buffer (10x Genomics, PN-2000153). The nuclei were stained by DAPI and trypan to check the quality and quantity. The isolated nuclei were processed immediately through the 10x Chromium Single Cell Platform according to the manufacturer's instructions (10x Genomics). Briefly, 16,000 resuspended nuclei (concentration 3,000-7,000 nuclei/μl) were combined with ATAC Buffer and ATAC Tn5 transposase (10x Genomics) to form a transposition mix. After incubation for 30 min at 37 °C, barcoding reagent and enzyme (10x Genomics) were added, and the resulting mix were loaded onto a single-cell chip, which was subsequently placed into a Chromium Single Cell Controller instrument for library construction. Quantification of DNA library was done by Agilent 2100 Bioanalyzer, then sequenced with an Illumina Hiseq2000 sequencer.