title: GSM5530550: Medicago truncatula roots' nuclei 24h after exposure to Ensifer meliloti, experimental replicate 1; Medicago truncatula; RNA-Seq
library_construction_protocol: After 24 hours of E. meliloti treatment, a 0.5-0.7-cm long root portions around the drop-inoculated spot were cut from 150 2-day-old M. truncatula seedlings. To perform nuclei isolation, root portions were placed on precooled glass plate on the top of an aluminum heating/cooling block kept on ice with 200 uL of NIB1 1× supplemented with 0.5 U/μL Protector RNase Inhibitor. Once all root segments were collected, they were chopped with a razor blade for 2 min. This step was repeated twice with a 30 sec interval in between. Then, chopped mixture was transferred to a 50 mL Falcon tube and nuclei were isolated. After the last wash step, the pellet was gently resuspended in a final volume of 750 μL of NIB2 1× + 0.2 U/μL Protector RNase Inhibitor using a Rainin 1 mL wide-bore tip and transferred to a 5 mL sterile tube compatible with the cell sorter. Nuclei were stained with 5 μg/ml DAPI for 5 min at room temperature and sorted using a BD FACSAria™ IIU/III upgraded cell sorter. 20k nuclei were used to generate each snRNA-seq library following the 10X Genomics Chromium Single Cell v3.1 protocol. The cDNA reaction was prepared as described previously. Two snRNA-seq libraries from two independent replicated experiments were generated.