GSM8389446: gt3a-KO, Mock, 24h, rep1; Arabidopsis thaliana; RNA-Seq
Fresh Nuclei purification buffer (NPB; 15mM Tris pH 7.5, 2mM EDTA, 80mM KCl, 20mM NaCl, 0.5mM spermidine, 0.2mM spermine, 1:100 BSA, and 1:100 protease inhibitor cocktail) was prepared before the experiment and chilled on ice. All the following procedures were performed on ice or at 4ºC. Twenty leaves were chopped in 500-1,000 µL of cold NPB with 1:500 Protector RNase IN with a razor blade on ice for 5 min to release nuclei and incubated in 20 mL of NPB. The crude nuclei extract was sequentially filtered through 70 µm and 30 µm cell strainers. Triton-X and NP40 were added to the extract at the final concentration of 0.1% each, and the extract was incubated at 4ºC for 5 min with rotation. The nuclei suspension was centrifuged at 50 x g for 3 min in a swing-rotor centrifuge to pellet non-nuclei debris, and the supernatant was taken. Nuclei were pelleted by centrifugation at 500 x g for 5 min with a swing-rotor centrifuge. When the pellet was green, the pellet was resuspended in 20 mL of NPBd (NPB, 0.1% Triton-X, and 0.1% NP40) with 1:1000 Protector RNase IN by pipetting, followed by centrifugation at 700 x g for 5 min. When the pellet was translucent, this step was skipped. The pellet was then washed by resuspending it in 20 mL of NPB with 1:1000 Protector RNase IN and centrifuging at 500 x g for 5 min with a swing-rotor centrifuge. The pellet was resuspended in 950 µL of 1X Nuclei Buffer (10X Genomics; PN-2000207) with 1:20 Protector RNase IN and 1mM DTT. The nuclei suspension was centrifuged at 50 x g for 3 min in a swing-rotor centrifuge to pellet non-nuclei debris, and the supernatant was taken. This step was repeated one more time. Then, nuclei were counted manually with a hemocytometer. Nuclei were pelleted by centrifugation at 500 x g for 5 min with a swing-rotor centrifuge and removed supernatant leaving approximately 10 µL of buffer. Nuclei were counted again, and up to 16k nuclei were used for the following steps. Single-cell RNA-seq and ATAC-seq libraries were constructed according to the manufacturer's instruction (10X Genomics, catalog: CG000338). Single-nucleus RNA-seq libraries were sequenced with Illumina NovaSeq 6000 in dual-indexmode with 10 cycles for i7 and i5 index. Single-nucleus ATAC-seq libraries were also sequenced with Illumina NovaSeq 6000 in dual-indexmode with eight and 24 cycles for i7 and i5 index, respectively. We followed the 10X genomics gene expression assay protocols