GSM8866528: young needles and buds; Taxus x media; RNA-Seq
Nuclei extraction buffer (NIB) consisted of 5 mM MgCl2, 10 mM HEPES pH 7.6, 0.8 M sucrose, 0.1% Triton X and (for density matching to prevent nuclei settling during flow sorting) 1% Dextran T40 and 2% Ficoll. On the day of use, NIB was supplemented with 1 mM dithiothreitol. All nuclei extraction steps were conducted at 4°C and wide bore pipette tips were used when handling nuclei. Steps between tissue harvesting and loading into the Chromium device were completed within 90 minutes to avoid RNA loss. To isolate nuclei, approximately 1 g of Taxus media tissue was removed from the plant and immediately placed in a petri dish with 10mL NIB. Tissue was chopped by hand at ~200 rpm with a fresh razor blade for 5 minutes until most large tissue was broken down, and then gently rocked at 4°C for 15 minutes. To remove large debris, disrupted tissue was then passed through a pre-wet 100 μm cell strainer stacked atop a 40 μm cell strainer. Nuclei were gently pelleted at 300 g at 4°C for 5 minutes and resuspended in 1 mL NIB with 5 ng/μL 4,6-diamidino-2-phenylindole (DAPI, Thermo Fischer) and 5 ng/μL propidium iodide. Using a Sony SH800 cell sorter with a 70 μm chip, 140,000-200,000 nuclei were sorted into a tube containing 1 mL PBS+ (PBS, 0.1% bovine serum albumin, 20 U/mL Invitrogen Ribonuclease Inhibitor).  Nuclei were centrifuged at 300g at 4°C for 5 minutes, then gently resuspended in 40 μL PBS+. Nuclei were immediately loaded onto a 10x Genomics Chromium controller and libraries were generated using v3 chemistry. Nuclei were immediately loaded onto a 10x Genomics Chromium controller and libraries were generated using v3 chemistry.  Libraries were sequenced on an Illumina NextSeq 3000.