GSM8482713: RNA_lib1_mbd567_rep2; Arabidopsis thaliana; RNA-Seq
Pollen and RNA extraction was performed following protocol in Ichino et al. 2022. Briefly, ~500 uL of open flowers were harvested into 1.7 mL epp tubes, then 700 uL of Galbraith buffer (45 mM MgCl2, 30 mM C6H5Na3O7.2H2O [Trisodium citrate dihydrate], 20 mM MOPS, 0.1% [v/v] Triton X-100, pH 7, 5 l per ml of 14.3M Beta-mercaptoethanol [CAS-60-24-2, Fisher Scientific]) was added and samples were vortexed at max strength 2 min at 4C to release pollen from anthers. The suspension was filtered with an 80 um nylon mesh into a new 1.5 ml tube. The procedure was repeated one more time with the same flowers to increase the yield of pollen. The two aliquots of filtered pollen suspension were combined and centrifuged for 5 min at 500 g, 4°C. The pollen pellet was frozen in liquid nitrogen and stored at -80C. This procedure was repeated on consecutive days to obtain replicates. Frozen flower bud or pollen samples were disrupted with a tissue grinder and RNA extraction was performed with the Zymo Direct-zol RNA MiniPrep kit (Zymo Research) with on-column DNase I digestion. RNA-seq libraries were generated using the TruSeq Stranded mRNA Library Prep Kit (Illumina), following the manufacturer's instructions and starting with 500-1000 ng of RNA as input and sequenced on a Novaseq 6000 machine.