GSM7035823: VASA_cambium, rep2, snRNAseq; Arabidopsis thaliana; RNA-Seq
Hypocotyls were dissected from Arabidopsis and collected on petri dishes kept on ice. 2ml of 1x nuclei isolation buffer (CelLytic ™ PN Isolation/Extraction Kit, Sigma #CELLYTPN1) supplemented with 20 µl RiboLock RNase inhibitor 40 U/µL (ThermoFisher #EO0381) and Hoechst 33342 at 10 µg/ml final concentration were prepared and minimum amount of buffer was applied to the hypocotyls to submerge the collected hypocotyls. Hypocotyls were chopped using razor blades (Wilkinson Sword) for several minutes (up to 5 min.) and transferred on a gentle shaker in at 4 degrees for 15 min. Samples were then filtered through a 50 µm filter (CellTrics, Sysmex #04-004-2327) and passed to a low protein binding tube (Eppendorf #0030108132). Nuclei were further purified in FANS with 405-nm laser, and collectively sorted into a tube for 10x Genomics application.  Single nuclei were sorted into the single well of 384-well plates for VASA-seq application with GFP and RFP signal information, where ERCC spike-in controls and well-specific barcodes are included in each well. O21-24, P21-24 wells (barcodes: "AGTTGGAC", "AGTGCAGA", "CTCTGTCT", "CTCTGCTA", "TAGTTGCG", "AGGCTAAC") were kept blank for quality control. 10x Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). FANS collected nuclei were applied without dilution, resulted in about 6,000 nuclei application. For VASA-seq, plates were processed according to the published VASA-plate protocol (doi: 10.1038/s41587-022-01361-8).