title: GSM3602404: WT_IP_2; Arabidopsis thaliana; miRNA-Seq
library_construction_protocol: AGO1-IP RNA extraction: 0.5g seedlings of Col and fry1-6 were ground in liquid nitrogen, the prowder was suspend with 1.5ml IP buffer containing 50mM Tris-Cl, pH7.5, 150mM NaCl, 0.1% NP40,  4mM MgCl2, 5mM DTT and 1x proteinase inhibitor cocktail (Roche). The slurry was clarified by 12000g centrifugation twice at 4℃. 4ul AGO1 antibody (Agrisera, AS09 527 ) was added into the supernatant  and incubate for 2h  at 4℃. Then 30ul Protein A Dynabeads (Invitrogen, 10002D) was added for 1h incubation at 4℃. IP products was washed by IP buffer for 4 times.  RNAs were isolated from these samples by Tri Reagent (Molecular Research Center). 20ug total RNA was resolved on 15% Ura PAGE, and15-40nt small RNAs were recovered by gel purification. The recovery small RNA products  were used for small RNA  library construction with NEBNext® Multiplex Small RNA Library kit (NEB, E7300S).
attributes.tag: GEO Accession
attributes.value: GSM3602404