GSM8153841: Ah20mirS2_AGO1-IP; Arabidopsis thaliana; ncRNA-Seq
AGO1 and AGO4 immunoprecipitation (IP) assays were performed on immature buds of plants carrying an homozygous Ah04mir1887, Ah20mirS3 or Ah20mirS2 transgene, using a protocol adapted from (Schott et al., 2012), with the following modifications: IPs were performed on 1.4 g of finely ground powder of frozen immature buds. The anti-AGO1 (AS09 527, Agrisera) and anti-AGO4 (AS09 617, Agrisera) antibodies were added at a 1/500 dilution for the incubation step and their immobilization was performed using Dynabeads Protein G (Invitrogen). The IP products were washed six times with PBS prior to sRNA extraction using the Trizol reagent (Ambion). In parallel, an aliquot of each input extract was used for total sRNA isolation using the Trizol LS reagent (Invitrogen), according to the supplier instructions. For each genotype three sRNA fractions were obtained: AGO1-IP, AGO4-IP and input. These three sRNA fractions were subjected to an acrylamide gel-based size selection for sRNAS. TruSeq Small RNA library kit (Illumina)