GSM5321860: X26 AG-domain RNA-seq; Arabidopsis thaliana; RNA-Seq
Nuclei were isolated by FANS from pAP1:AP1-GR ap1-1 cal-1 transgenic plants expressing a GFP labeled nuclei envelope protein driven by tissue-specific promoters. We used AP3 promote and AG 2nd intron plus a minimal 35 s promoter element as promoters for the constructs to mark AP3 and AG expressing domains in flowers, respectively. After nuclei isolation, as described in the previous paragraph, nuclei were sorted into a 1.5 tube with 15μl of 4% BSA in PBS and 6μl of RiboLock RNase Inhibitor by a BD FACS Aria III. The GFP channel was set using pAP1:AP1-GR ap1-1 cal-1 as a negative control, and then nuclei were selected by gating on the DAPI peaks under the GFP positive events. After sorting, nuclei were pelleted at 1500 rcf for 10mins at 4°C, and the supernatant was then removed. Nuclei were lysed by vortex in 350 μl RLT buffer with 2-Mercaptoethanol, and RNA was then isolated from sorted nuclei with Qiagen RNeasy Micro Kit. cDNA synthesis was done with SMART-Seq® v4 Ultra® Low Input RNA Kit following the manufacturer's instructions. cDNA was sheared to 200–500 bp size by Covaris AFA system and constructed with sequencing adaptors by ThruPLEX DNA-Seq Kit.ThruPLEX DNA-Seq Kit.