GSM5733049: snRNA-seq_day3 [stage 4]; Arabidopsis thaliana; RNA-Seq
Inflorescences were gently crushed to pieces in liquid nitrogen using a mortar and a pestle and then transferred to a gentleMACS M tube. After liquid nitrogen evaporated totally, 5 ml of Honda buffer (2.5 % Ficoll 400, 5 % Dextran T40, 0.4 M sucrose, 10 mM MgCl2, 1 μM DTT, 0.5% Triton X-100, 1 tablet/50 ml cOmplete Protease Inhibitor Cocktail, 0.4 U/μl RiboLock, 25 mM Tris-HCl, pH 7.4) was added to the tube. Nuclei were released at 4 °C by homogenizing the tissue on a gentleMACS Dissociator with a running program as described previously. The resulting homogenate was filtered through a 70 μm strainer, and another 5ml Honda buffer was applied onto the filter to collect the remaining nuclei. Nuclei were then pelleted by centrifugation at 1000 g for 6 min at 4 °C and then resuspended gently in 500 μl Honda buffer. The nuclei suspension was filtered again through a 30 μm strainer, diluted by adding 500ul PBS buffer, and stained with 2 μM DAPI. Ambion RNase Inhibitor and SUPERaseIn RNase Inhibitor were added to a final concentration of 0.4 U/μl and 0.2 U/μl, respectively. 200,000 events of single nuclei were selected on DAPI signals by a BD FACS Aria Fusion into an 1.5ml tube with landing buffer (15μl 4% BSA in PBS with 80U Ambion RNase Inhibitor and 80U SUPERaseIn RNase Inhibitor). Sorted nuclei were counted in Neubauer counting chambers under a Leica DMi8 fluorescent microscope. Single-nuclei RNA-seq library was prepared from 10,000 freshly-isolated plant nuclei with the Chromium Single Cell 3ʹ Reagent Kits v3 according to the manufacturer's instructions. 11 PCR cycles were used for cDNA amplification, and 14 PCR cycles were used for final amplification of the constructed libraries. The average fragment size of the snRNA-seq library was checked with an Agilent High Sensitivity D1000 ScreenTape, and the concentration was measured with Qubit 1X dsDNA HS Assay Kit.