sRNA-seq of Arabidopsis halleri Auby: root_input
The Argonaute immunoprecipitations have been done according to Barre-Villeneuve et al. (2024) with anti-AGO1 antibodies (AS09 527, Agrisera) and Anti-AGO4 antibodies (AS09 617, Agrisera). Tissues from pooled individuals were ground in liquid nitrogen and were homogenized in extraction buffer EB (50 mM Tris-HCl at pH 7.5, 150 mM NaCl, 5 mM MgCl2, 0.2% v/v NP40, 10% glycerol, 10 M MG132) containing the EDTA-free protease inhibitor cocktail (Roche). After 15 min of incubation at 4C, cell debris were removed by centrifugation at 21,000g for 30 min at 4C. The clarified lysate was incubated for 2 h at 4C at 7-10 rpm, with AGO1 or AGO4 antibodies (from agrisera), and then 1 h at 4C at 7-10 rpm with dynabeads protein A (Invitrogen), equilibrated with the EB . Beads were isolated using a magnetic rack, and washed once with 1 mL of EB and 4 times with 1 mL of PBS (Gibco). The sRNA were extracted from total/inputs and immunoprecipitated fractions using respectively Trizol and Trizol-LS, according to supplier instructions (Invitrogen). Subsequent sRNA libraries were performed and sequenced by the POPS platform from the plants science institute of Paris-Saclay (IPS2).