Arabidopsis thaliana
Protocols: Seedlings and fully developed flowers from wild type plants were collected and snap-frozen in liquid nitrogen. Plants were grown under standard conditions. The frozen tissue was carefully crushed to small pieces in liquid nitrogen using a mortar and pestle. The frozen tissue pieces were then added to a gentleMACS C tube together with 5 ml of Honda buffer (2.5 % Ficoll 400, 5 % Dextran T40, 0.4 M sucrose, 10 mM MgCl2 , 1µM DTT, 0.5% Triton X-100, 1 tablet/50mL cOmplete Protease Inhibitor Cocktail, 0.4 U/µl RiboLock, 25 mM Tris-HCl, pH 7.4). The C tubes were put onto a gentleMACS Dissociator and the program “arabidopsis1” (Supplemental Table X) was run at 4°C to disrupt the tissue and release the nuclei. The resulting suspension was then filtered through a 70 µm strainer and centrifuged at 1000 g for 6 min at 4°C. The pellet was resuspended carefully in 500 µl Honda buffer, filtered through a 35 µm strainer and stained with 3x staining buffer (12 µM DAPI, 0.4 U/µl Ambion RNase Inhibitor, 0.2 U/µl SUPERaseIn RNase Inhibitor in PBS). Single nuclei were sorted by gating on the DAPI peaks with a BD FACS Aria III (200,000 – 400,000 events) into a small volume of landing buffer (4% BSA in PBS, 2 U/µl Ambion RNase Inhibitor, 1 U/µl SUPERaseIn RNase Inhibitor). Sorted nuclei were additionally stained with NucBlue from the Invitrogen Ready Probes Cell Viability Imaging Kit (Blue/Red), then counted and checked for integrity in Neubauer counting chambers. The NucBlue and DAPI co-stained single nuclei suspension (60 cells/µL) was distributed to eight wells of a 384-well source plate (Cat. No. 640018, Takara) and then dispensed into a barcoded SMARTer ICELL8 3' DE Chip (Cat. No. 640143, Takara) by an ICELL8 MultiSample NanoDispenser (MSND, Takara). Chips were centrifuged at 500 g for 5 min at 4°C. Nanowells were imaged using the ICELL8 Imaging Station (Takara). After imaging, the chip was sealed, placed in a pre-cooled freezing chamber, and stored at −80 °C for at least 2 hrs. The CellSelect software was used to identify nanowells that contained a single nucleus. One chip yielded 800 - 1200 single-nucleus-nanowells. These nanowells were selected for subsequent targeted deposition of 50 nL/nanowell RT-PCR reaction mix from the SMARTer ICELL8 3' DE Reagent Kit (Cat. No. 640167, Takara) using the MSND. After RT and amplification in a Chip Cycler, barcoded cDNA products from nanowells were pooled by means of the the SMARTer ICELL8 Collection Kit (Cat. No. 640048, Takara). cDNA was concentrated using the Zymo DNA Clean & Concentrator kit (Cat. No. D4013, Zymo Research) and purified with AMPure XP beads. Afterwards, cDNA was used to construct Nextera XT (Illumina) DNA libraries followed by AMPure XP bead purification. Qubit dsDNA HS Assay Kit, KAPA Library Quantification Kit for Illumina Platforms and Agilent HighSensitivity D1000 ScreenTape Assay were used for library quantification and quality assessment.
isolate
not applicable
organism
Arabidopsis thaliana
cultivar
Co-0
age
28
genotype
wild type genotype
developmental stage
mixed flower stages before flower stage 13
organism part
inflorescence except open flowers
cell_type
plant cell
ENA-FIRST-PUBLIC
2020-10-01
ENA-LAST-UPDATE
2021-08-09