GSM7063666: 0-day-old imbibed seeds, run 3, snRNA-seq; Arabidopsis thaliana; RNA-Seq
Tissue was chopped in 500-1,000 µL of cold NPB (NPB; 20mM MOPS (pH 7), 40mM NaCl, 90mM KCl, 2mM EDTA, 0.5mM EGTA, 0.5mM spermidine, 0.2mM spermine, 1:100 protease inhibitor cocktail, 2% BSA, and 1:1000 SUPERase IN) with a razor blade on ice for 5 min to release nuclei and incubated in 10 mL NPB. For the 0d and 1.25d seed samples, the nuclei were extracted in 5 mL NPB with a Dounce homogenizer with 20 strokes of the loose pestle followed by 20 strokes of the tight pestle. The crude nuclei extract was sequentially filtered through 70 µm and 30 µm cell strainers. BSA, Triton X-100, and NP40 were added to final concentrations of 2%, 0.05%, and 0.05%, respectively, and the extract was incubated at 4ºC for 10 min with rotation. Nuclei were pelleted by centrifugation at 700 x g for 5 min with a swing-rotor centrifuge, The pellet was resuspended in 10 mL of NPBd (NPB, 0.05% Triton X-100, 0.05% NP40, 2% BSA) by pipetting and incubated for 10 min, followed by centrifugation at 700 x g for 5 min, with additional washes until the pellet became translucent. The pellet was then resuspended in 1 mL NPB and centrifuged at 50 x g for 3 min in a fixed-rotor centrifuge to pellet non-nuclei debris (including pollen grains from flower tissues), and the supernatant was taken. This step was repeated until the supernatant cleared or a significant size of pellet was not observed. Finally, nuclei were pelleted by centrifugation at 700 x g for 10 min with a swing-rotor centrifuge and resuspended in an appropriate volume of NPB. Nuclei were counted manually with a hemocytometer. Library construction was performed according to the manufacturer's specification (Chromium Next GEM Single Cell 3' HT Kit v3.1, 10x Genomics)