GSM5608793: small RNA data of DCL3 H909A rep2 in dcl234; Arabidopsis thaliana; ncRNA-Seq
Floral tissues from 5-6 week old seedlings were used for RNA extraction with Zymo Direct-zol RNA MiniPrep kit (Zymo Research) following manufacturer's instructions. Floral tissues of two independent T1 plants of each genotype were used to characterize the abundance of 24 nt siRNA. Total RNA was extracted using the Direct-zol RNA Miniprep kit (Zymo) according to the manufacturer's instructions. Two micrograms of total RNA was mixed with equal volume of the 2x RNA loading dye (NEB), denatured at 65◦C for 10 min and immediately chilled on ice. Denatured total RNA was separated on 15% TBE urea gel (Invitrogen) and small RNAs from 15-30 nts were excised. The excised gel pieces were forced through gel breaker tubes (IST engineering inc) by centrifuging at 20,000 g for 3 min. small RNA was then eluted with nuclease free water by incubation at 70◦C for 10 min and precipitated with EtOH. Small RNA libraries were made using NEBNext® Small RNA Library Prep Set for Illumina® (Multiplex Compatible) (NEB) according to the manufacturer's instructions.