GSM7063695: 44-day-old silique, run 8, 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. 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)